Modified oligonucleotides with increased stability

ABSTRACT

This disclosure relates to novel modified oligonucleotides. Novel modified siRNA are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication Ser. Nos. 62/824,136, filed Mar. 26, 2019; 62/826,454, filedMar. 29, 2019, and 62/864,792, filed Jun. 21, 2019, the entiredisclosures of each of which are hereby incorporated herein byreference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant numbersNS104022 and OD020012 awarded by the National Institutes of Health. TheGovernment has certain rights in the invention.

FIELD OF THE INVENTION

This disclosure relates to novel modified oligonucleotides, and novelmodified siRNA.

BACKGROUND

Currently, the most common metabolically stable backbone modificationused in complex therapeutic RNAs is the Phosphorothioate (PS)modification. While other available backbone modification alternatives,such as Peptide Nucleic Acid (PNA) and Phosphorodiamidate MorpholinoOligonucleotide (PMO), work well as steric-blocking antisenseoligonucleotides, they are not tolerated in many promising RNA-basedtherapeutic strategies. These strategies include siRNAs, miRNAs,RNaseH-dependent antisense oligonucleotides, aptamer-based therapeutics,and CRISPR therapeutics. This poor tolerance is due to PNA's and PMO'sinability to withstand biological machineries, such as Argonauteproteins (siRNA/miRNA), Cas9 (CRISPR) and RNaseH that strictly recognizeRNA structures when they form “functional” RNA-protein complexes.

One of the most common RNA-based therapeutic strategies is the use ofmetabolically stable, PS-modified RNAs. One severe drawback in thisstrategy, however, is toxicity due to non-specific binding of the RNAsto a variety of proteins in vivo. Thus, additional metabolically stablebackbone modifications are urgently needed in the field of RNAtherapeutics.

Provided herein are a variety of backbone modifications wherein thebridging oxygen of the phosphodiester bond is substituted with variousorganic functional groups. The backbone modifications provided hereinare not expected to have a profound impact on the structure of RNA, andcan therefore provide compatibility with a variety of RNA-bindingbiological machineries. Further, these modifications are not expected todisplay toxic, non-specific binding to proteins, and thus can beincorporated into a wide range of therapeutic RNAs.

SUMMARY

In one aspect, the disclosure provides a modified oligonucleotide, saidoligonucleotide having a 5′ end, a 3′ end, that is complementary to atarget, wherein the oligonucleotide comprises a sense and antisensestrand, and at least one modified intersubunit linkage of Formula I:

wherein:

each B is, independently, a base pairing moiety;

W is selected from the group consisting of O, OCH₂, OCH, CH₂, and CH,optionally wherein W is selected from the group consisting of OCH₂ andOCH;

each X is, independently, selected from the group consisting of halo(e.g., fluoro or chloro), hydroxy, and C₁₋₆ alkoxy, optionally whereineach X is, independently, selected from the group consisting of halo(e.g., fluoro or chloro) and C₁₋₆ alkoxy (e.g., methoxy, ethoxy,n-propoxy, sec-propoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, orn-heptoxy);

Y is selected from the group consisting of O⁻, OH, OR, NH⁻, NH₂, S⁻, andSH, optionally wherein Y is selected from the group consisting of O⁻,OH, and OR. Z is selected from the group consisting of O and CH₂;

R is a protecting group; and

is an optional double bond.

In some embodiments of Formula I, W is OCH₂.

In some embodiments of Formula I, W is OCH and

is a double bond.

In some embodiments of Formula I, Z is O.

In some embodiments of Formula I, Z is CH₂.

In some embodiments of Formula I, when Y is O⁻, either Z or W is not O.

In some embodiments of Formula I, Z is CH₂ and W is CH₂. In someembodiments, the modified intersubunit linkage of Formula I is amodified intersubunit linkage of Formula II:

In some embodiments of Formula I, Z is CH₂ and W is O. In someembodiments, the modified intersubunit linkage of Formula I is amodified intersubunit linkage of Formula III:

In some embodiments of Formula I, Z is O and W is CH₂. In someembodiments, the modified intersubunit linkage of Formula I is amodified intersubunit linkage of Formula IV:

In some embodiments of Formula I, Z is O and W is CH. In someembodiments, the modified intersubunit linkage of Formula I is amodified intersubunit linkage of Formula V:

In some embodiments, the modified intersubunit linkage of Formula I is amodified intersubunit linkage of Formula VI:

In some embodiments of Formula VI:

-   -   each B is, independently, a base pairing moiety;    -   each X is, independently, selected from the group consisting of        halo, hydroxy, and C₁₋₆ alkoxy; optionally wherein each X is,        independently, selected from the group consisting of halo (e.g.,        fluoro) and C₁₋₆ alkoxy (e.g., methoxy, ethoxy, n-propoxy,        sec-propoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, or        n-heptoxy);    -   Y is selected from the group consisting of O⁻, OH, OR, NH⁻, NH₂,        S⁻, and SH, optionally wherein Y is selected from the group        consisting of O⁻, OH, and OR;    -   Z is selected from the group consisting of O and CH₂; and    -   is an optional double bond.

In some embodiments of Formula VI:

-   -   each X is, independently, selected from the group consisting of        fluoro, hydroxy, and C₁₋₆ alkoxy; optionally wherein each X is,        independently, selected from the group consisting of fluoro and        C₁₋₆ alkoxy (e.g., methoxy, ethoxy, n-propoxy, sec-propoxy,        n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, or n-heptoxy);    -   Y is selected from the group consisting of O⁻, OH, and OR;    -   Z is selected from the group consisting of O and CH₂; and    -   is an optional double bond.

In some embodiments of Formula VI:

-   -   each X is, independently, selected from the group consisting of        fluoro, hydroxy, methoxy, ethoxy, n-propoxy, sec-propoxy,        n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, and n-heptoxy;    -   Y is selected from the group consisting of O⁻, OH, and OR;    -   Z is selected from the group consisting of O and CH₂; and    -   is an optional double bond.

In some embodiments of Formula VI:

-   -   each X is, independently, selected from the group consisting of        fluoro, hydroxy, methoxy, ethoxy, n-propoxy, sec-propoxy,        n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, and n-heptoxy;    -   Y is selected from the group consisting of O⁻, OH, and OR;    -   Z is O; and    -   is an optional double bond.

In some embodiments of Formula VI:

-   -   each X is, independently, selected from the group consisting of        fluoro, hydroxy, methoxy, ethoxy, n-propoxy, sec-propoxy,        n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, and n-heptoxy;    -   Y is selected from the group consisting of O⁻, OH, and OR;    -   Z is CH₂; and    -   is an optional double bond.

In some embodiments of Formula VI:

-   -   each X is, independently, selected from the group consisting of        fluoro, hydroxy, and methoxy;    -   Y is selected from the group consisting of O⁻, OH, and OR;    -   Z is O; and    -   is an optional double bond.

In some embodiments of Formula VI:

-   -   each X is, independently, selected from the group consisting of        fluoro, hydroxy, and methoxy;    -   Y is selected from the group consisting of O⁻, OH, and OR;    -   Z is CH₂; and    -   is an optional double bond.

In some embodiments of Formula I, Z is O and W is OCH₂. In someembodiments, the modified intersubunit linkage of Formula I is amodified intersubunit linkage of Formula VIa:

In some embodiments of Formula I, Z is CH₂ and W is CH. In someembodiments, the modified intersubunit linkage of Formula I is amodified intersubunit linkage of Formula VII:

In some embodiments of Formula I, the base pairing moiety B is selectedfrom the group consisting of adenine, guanine, cytosine, and uracil.

In some embodiments, the modified oligonucleotide is incorporated intosiRNA, said modified siRNA having a 5′ end, a 3′ end, that iscomplementary to a target, wherein the siRNA comprises a sense andantisense strand, and at least one modified intersubunit linkage ofFormula I:

wherein:

each B is, independently, a base pairing moiety;

W is selected from the group consisting of O, OCH₂, OCH, CH₂, and CHoptionally wherein W is selected from the group consisting of OCH₂ andOCH;

each X is, independently, selected from the group consisting of halo(e.g., fluoro or chloro), hydroxy, and C₁₋₆ alkoxy, optionally whereineach X is, independently, selected from the group consisting of halo andC₁₋₆ alkoxy (e.g., methoxy, ethoxy, n-propoxy, sec-propoxy, n-butoxy,sec-butoxy, tert-butoxy, n-pentoxy, or n-heptoxy);

Y is selected from the group consisting of O⁻, OH, OR, NH⁻, NH₂, S⁻, andSH, optionally wherein Y is selected from the group consisting of O⁻,OH, and OR;

Z is selected from the group consisting of O and CH₂;

R is a protecting group; and

is an optional double bond.

In some embodiments of Formula I, when Y is O⁻, either Z or W is not O.In some embodiments of Formula I, Z is CH₂ and W is CH₂.

In some embodiments, the modified intersubunit linkage of Formula I is amodified intersubunit linkage of Formula II:

In some embodiments of Formula I, Z is CH₂ and W is O. In someembodiments, the modified intersubunit linkage of Formula I is amodified intersubunit linkage of Formula III:

In some embodiments of Formula I, Z is O and W is CH₂. In someembodiments, the modified intersubunit linkage of Formula I is amodified intersubunit linkage of Formula IV:

In some embodiments of Formula I, Z is O and W is CH. In someembodiments, the modified intersubunit linkage of Formula I is amodified intersubunit linkage of Formula V:

In an embodiment of Formula I, wherein Z is CH═CH and W is CH₂. Inanother embodiment, the modified intersubunit linkage of Formula I is amodified intersubunit linkage of Formula VII:

In some embodiments, the modified intersubunit linkage of Formula I is amodified intersubunit linkage of Formula VI:

In some embodiments of Formula VI:

-   -   each B is, independently, a base pairing moiety;    -   each X is, independently, selected from the group consisting of        halo, hydroxy, and C₁₋₆ alkoxy; optionally wherein each X is,        independently, selected from the group consisting of halo (e.g.,        fluoro) and C₁₋₆ alkoxy (e.g., methoxy, ethoxy, n-propoxy,        sec-propoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, or        n-heptoxy);    -   Y is selected from the group consisting of O⁻, OH, OR, NH⁻, NH₂,        S⁻, and SH, optionally wherein Y is selected from the group        consisting of O⁻, OH, and OR;    -   Z is selected from the group consisting of O and CH₂; and    -   is an optional double bond.

In some embodiments of Formula VI:

-   -   each X is, independently, selected from the group consisting of        fluoro, hydroxy, and C₁₋₆ alkoxy; optionally wherein each X is,        independently, selected from the group consisting of fluoro and        C₁₋₆ alkoxy (e.g., methoxy, ethoxy, n-propoxy, sec-propoxy,        n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, or n-heptoxy);    -   Y is selected from the group consisting of O⁻, OH, and OR;    -   Z is selected from the group consisting of O and CH₂; and    -   is an optional double bond.

In some embodiments of Formula VI:

-   -   each X is, independently, selected from the group consisting of        fluoro, hydroxy, methoxy, ethoxy, n-propoxy, sec-propoxy,        n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, and n-heptoxy;    -   Y is selected from the group consisting of O⁻, OH, and OR;    -   Z is selected from the group consisting of O and CH₂; and    -   is an optional double bond.

In some embodiments of Formula VI:

-   -   each X is, independently, selected from the group consisting of        fluoro, hydroxy, methoxy, ethoxy, n-propoxy, sec-propoxy,        n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, and n-heptoxy;    -   Y is selected from the group consisting of O⁻, OH, and OR;    -   Z is O; and    -   is an optional double bond.

In some embodiments of Formula VI:

-   -   each X is, independently, selected from the group consisting of        fluoro, hydroxy, methoxy, ethoxy, n-propoxy, sec-propoxy,        n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, and n-heptoxy;    -   Y is selected from the group consisting of O⁻, OH, and OR;    -   Z is CH₂; and    -   is an optional double bond.

In some embodiments of Formula VI:

-   -   each X is, independently, selected from the group consisting of        fluoro, hydroxy, and methoxy;    -   Y is selected from the group consisting of O⁻, OH, and OR;    -   Z is O; and    -   is an optional double bond.

In some embodiments of Formula VI:

-   -   each X is, independently, selected from the group consisting of        fluoro, hydroxy, and methoxy;    -   Y is selected from the group consisting of O⁻, OH, and OR;    -   Z is CH₂; and    -   is an optional double bond.

In some embodiments of Formula I, Z is O and W is OCH₂. In someembodiments, the modified intersubunit linkage of Formula I is amodified intersubunit linkage of Formula VIa:

In some embodiments of Formula I, wherein Z is CH₂ and W is CH. In someembodiments, the modified intersubunit linkage of Formula I is amodified intersubunit linkage of Formula VII:

In some embodiments, the base pairing moiety B is selected from thegroup consisting of adenine, guanine, cytosine, and uracil. In anotherembodiment, B is adenine. In yet another embodiment, B is guanine. Instill another embodiment, B is cytosine. In some embodiments, B isuracil.

In some embodiments, the modified oligonucleotide is incorporated intosiRNA, said modified siRNA having a 5′ end, a 3′ end, that iscomplementary to a target, wherein the siRNA comprises a sense andantisense strand, and at least one modified intersubunit linkage ofFormula I:

wherein:

each B is, independently, a base pairing moiety;

W is selected from the group consisting of O, OCH₂, OCH, CH₂, and CH,optionally wherein W is selected from the group consisting of OCH₂ andOCH;

each X is, independently, selected from the group consisting of halo(e.g., fluoro or chloro), hydroxy, and C₁₋₆ alkoxy, optionally whereineach X is, independently, selected from the group consisting of halo andC₁₋₆ alkoxy (e.g., methoxy, ethoxy, n-propoxy, sec-propoxy, n-butoxy,sec-butoxy, tert-butoxy, n-pentoxy, or n-heptoxy);

Y is selected from the group consisting of O⁻, OH, OR, NH⁻, NH₂, S⁻, andSH, optionally wherein Y is selected from the group consisting of O⁻,OH, and OR;

Z is selected from the group consisting of O and CH₂;

R is a protecting group selected from the group consisting ofdimethoxytrityl (DMTr), succinate, tert-butyl dimethylsilyl (TBDMS),benzoyl (Bz), benzyl (Bn), methoxyethoxymethyl ether (MOM),methoxybenzyl ether (PMB), methylthiomethyl ether, pivaloyl (Piv),tetrahydropyranyl (THP), tetrahydrofuranyl (THF), trityl (Trt),triisopropylsilyl (TIPS), tert-butyldiphenylsilyl (TBDPS), and acetate;and

is an optional double bond.

In some embodiments of Formula I, W is OCH₂.

In some embodiments of Formula I, W is OCH and

is a double bond;

In some embodiments of Formula I, Z is O.

In some embodiments of Formula I, Z is CH₂.

In some embodiments, the modified oligonucleotide is incorporated intosiRNA, said modified siRNA having a 5′ end, a 3′ end, that iscomplementary to a target and comprises a sense and antisense strand,wherein the siRNA comprises at least one modified intersubunit linkageis of Formula VIII:

wherein:

D is selected from the group consisting of O, OCH₂, OCH, CH₂, and CH,optionally wherein D is selected from the group consisting of OCH₂ andOCH;

C is selected from the group consisting of O⁻, OH, OR¹, NH⁻, NH₂, S⁻,and SH, optionally wherein C is selected from the group consisting ofO⁻, OH, and OR¹;

A is selected from the group consisting of O and CH₂;

R¹ is a protecting group;

is an optional double bond; and

the intersubunit is bridging two optionally modified nucleosides.

In some embodiments, D is OCH₂.

In some embodiments, D is OCH and

is a double bond.

In some embodiments, A is O.

In some embodiments, A is CH₂.

In some embodiments, when C is O⁻, either A or D is not O.

In some embodiments, D is CH₂. In another embodiment, the modifiedintersubunit linkage of Formula VIII is a modified intersubunit linkageof Formula IX:

In some embodiments, D is O. In another embodiment, the modifiedintersubunit linkage of Formula VIII is a modified intersubunit linkageof Formula X:

In some embodiments, D is CH₂. In another embodiment, the modifiedintersubunit linkage of Formula VIII is a modified intersubunit linkageof Formula XI:

In some embodiments, D is CH. In another embodiment, the modifiedintersubunit linkage of Formula VIII is a modified intersubunit linkageof Formula XII:

In another embodiment, the modified intersubunit linkage of Formula VIIis a modified intersubunit linkage of Formula XIV:

In some embodiments, D is OCH₂. In another embodiment, the modifiedintersubunit linkage of Formula VII is a modified intersubunit linkageof Formula XIII:

In another embodiment, the modified intersubunit linkage of Formula VIIis a modified intersubunit linkage of Formula XXa:

In some embodiments of the modified siRNA linkage, each optionallymodified nucleoside is independently, at each occurrence, selected fromthe group consisting of adenosine, guanosine, cytidine, and uridine.

In some embodiments, the modified oligonucleotide is incorporated intosiRNA, said modified siRNA having a 5′ end, a 3′ end, that iscomplementary to a target and comprises a sense and antisense strand,wherein the siRNA comprises at least one modified intersubunit linkageis of Formula VIII:

wherein:

D is selected from the group consisting of O, OCH₂, OCH, CH₂, and CH,optionally wherein D is selected from the group consisting of OCH₂ andOCH;

C is selected from the group consisting of O, OH, OR¹, NH⁻, NH₂, S⁻, andSH, optionally wherein C is selected from the group consisting of O⁻,OH, and OR¹;

A is selected from the group consisting of O and CH₂;

R¹ is a protecting group selected from the group consisting ofdimethoxytrityl (DMTr), succinate, tert-butyl dimethylsilyl (TBDMS),benzoyl (Bz), benzyl (Bn), methoxyethoxymethyl ether (MOM),methoxybenzyl ether (PMB), methylthiomethyl ether, pivaloyl (Piv),tetrahydropyranyl (THP), tetrahydrofuranyl (THF), trityl (Trt),triisopropylsilyl (TIPS), tert-butyldiphenylsilyl (TBDPS), and acetate;

is an optional double bond; and the intersubunit is bridging twooptionally modified nucleosides.

In some embodiments, D is OCH₂.

In some embodiments, D is OCH and

is a double bond.

In some embodiments, A is O.

In some embodiments, A is CH₂.

In one aspect, the disclosure provides a method of treating or managinga neurodegenerative disease comprising administering to a patient inneed of such treatment or management a therapeutically effective amountof the siRNA or modified oligonucleotide recited above.

In some embodiments, the siRNA or modified oligonucleotide isadministered to the brain of the patient.

In some embodiments, the siRNA or modified oligonucleotide isadministered by intracerebroventricular (ICV) injection.

In one aspect, there is provided a branched compound comprising two ormore oligonucleotides, wherein: (a) the oligonucleotides are connectedto one another by one or more moieties selected from a linker, a spacerand a branching point, and (b) at least one oligonucleotide is amodified oligonucleotide comprising at least one modified intersubunitlinkage of Formula (I):

wherein:

each B is, independently, a base pairing moiety;

W is selected from the group consisting of O, OCH₂, OCH, CH₂, and CH,optionally wherein W is selected from the group consisting of OCH₂ andOCH;

each X is, independently, selected from the group consisting of halo(e.g., fluoro or chloro), hydroxy, and C₁₋₆ alkoxy, optionally whereineach X is, independently, selected from the group consisting of halo andC₁₋₆ alkoxy (e.g., methoxy, ethoxy, n-propoxy, sec-propoxy, n-butoxy,sec-butoxy, tert-butoxy, n-pentoxy, or n-heptoxy);

Y is selected from the group consisting of O⁻, OH, OR, NH⁻, NH₂, S⁻, andSH, optionally wherein Y is selected from the group consisting of O⁻,OH, and OR.

Z is selected from the group consisting of O and CH₂;

R is a protecting group; and

is an optional double bond.

In some embodiments, W is OCH and

is a double bond.

In some embodiments, Z is O.

In some embodiments, Z is CH₂.

In some embodiments, the branched compound comprises 2, 4, 6, or 8oligonucleotides.

In some embodiments, each oligonucleotide is double-stranded andcomprises a sense strand and an antisense strand, wherein the sensestrand and the antisense strand each have a 5′ end and a 3′ end.

In some embodiments, each double-stranded oligonucleotide isindependently connected to a linker, spacer or branching point at the 3′end or at the 5′ end of the sense strand or the antisense strand.

In some embodiments, each antisense strand independently comprises atleast 16, at least 17, at least 18, at least 19, or at least 20contiguous nucleotides, and has complementarity to a target.

In some embodiments, each linker is independently selected from anethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate,a phosphonate, a phosphoramidate, an ester, an amide, a triazole, andcombinations thereof, wherein any carbon or oxygen atom of the linker isoptionally replaced with a nitrogen atom, bears a hydroxyl substituent,or bears an oxo substituent.

In some embodiments, when Y is O⁻, either Z or W is not O.

In some embodiments, Z is CH₂ and W is CH₂.

In some embodiments, the modified intersubunit linkage of Formula (I) isa modified intersubunit linkage of Formula (II):

In some embodiments, Z is CH₂ and W is O.

In some embodiments, the modified intersubunit linkage of Formula (I) isa modified intersubunit linkage of Formula (III):

In some embodiments, Z is O and W is CH₂.

In some embodiments, the modified intersubunit linkage of Formula (I) isa modified intersubunit linkage of Formula (IV):

In some embodiments, Z is O and W is CH.

In some embodiments, the modified intersubunit linkage of Formula (I) isa modified intersubunit linkage of Formula (V):

In some embodiments, Z is O and W is OCH₂.

In some embodiments, the modified intersubunit linkage of Formula I is amodified intersubunit linkage of Formula VI:

In some embodiments of Formula VI:

-   -   each B is, independently, a base pairing moiety;    -   each X is, independently, selected from the group consisting of        halo, hydroxy, and C₁₋₆ alkoxy; optionally wherein each X is,        independently, selected from the group consisting of halo (e.g.,        fluoro) and C₁₋₆ alkoxy (e.g., methoxy, ethoxy, n-propoxy,        sec-propoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, or        n-heptoxy);    -   Y is selected from the group consisting of O⁻, OH, OR, NH⁻, NH₂,        S⁻, and SH, optionally wherein Y is selected from the group        consisting of O⁻, OH, and OR;    -   Z is selected from the group consisting of O and CH₂; and    -   is an optional double bond.

In some embodiments of Formula VI:

-   -   each X is, independently, selected from the group consisting of        fluoro, hydroxy, and C₁₋₆ alkoxy; optionally wherein each X is,        independently, selected from the group consisting of fluoro and        C₁₋₆ alkoxy (e.g., methoxy, ethoxy, n-propoxy, sec-propoxy,        n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, or n-heptoxy);    -   Y is selected from the group consisting of O⁻, OH, and OR;    -   Z is selected from the group consisting of O and CH₂; and    -   is an optional double bond.

In some embodiments of Formula VI:

-   -   each X is, independently, selected from the group consisting of        fluoro, hydroxy, methoxy, ethoxy, n-propoxy, sec-propoxy,        n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, and n-heptoxy;    -   Y is selected from the group consisting of O⁻, OH, and OR;    -   Z is selected from the group consisting of O and CH₂; and    -   is an optional double bond.

In some embodiments of Formula VI:

-   -   each X is, independently, selected from the group consisting of        fluoro, hydroxy, methoxy, ethoxy, n-propoxy, sec-propoxy,        n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, and n-heptoxy;    -   Y is selected from the group consisting of O⁻, OH, and OR;    -   Z is O; and    -   is an optional double bond.

In some embodiments of Formula VI:

-   -   each X is, independently, selected from the group consisting of        fluoro, hydroxy, methoxy, ethoxy, n-propoxy, sec-propoxy,        n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, and n-heptoxy;    -   Y is selected from the group consisting of O⁻, OH, and OR;    -   Z is CH₂; and    -   is an optional double bond.

In some embodiments of Formula VI:

-   -   each X is, independently, selected from the group consisting of        fluoro, hydroxy, and methoxy;    -   Y is selected from the group consisting of O⁻, OH, and OR;    -   Z is O and    -   is an optional double bond.

In some embodiments of Formula VI:

-   -   each X is, independently, selected from the group consisting of        fluoro, hydroxy, and methoxy;    -   Y is selected from the group consisting of O⁻, OH, and OR;    -   Z is CH₂; and    -   is an optional double bond.

In some embodiments, the modified intersubunit linkage of Formula (I) isa modified intersubunit linkage of Formula (VIa):

In some embodiments, Z is CH₂ and W is CH.

In some embodiments, the modified intersubunit linkage of Formula (I) isa modified intersubunit linkage of Formula (VII):

In some embodiments, wherein the base pairing moiety B is selected fromthe group consisting of adenine, guanine, cytosine, and uracil.

In some embodiments, the modified oligonucleotide is incorporated into amodified siRNA, said modified siRNA having a 5′ end, a 3′ end, that iscomplementary to a target, wherein the siRNA comprises a sense andantisense strand, and at least one modified intersubunit linkage ofFormula (I).

In some embodiments, R is a protecting group selected from the groupconsisting of dimethoxytrityl (DMTr), succinate, tert-butyldimethylsilyl (TBDMS), benzoyl (Bz), benzyl (Bn), methoxyethoxymethylether (MOM), methoxybenzyl ether (PMB), methylthiomethyl ether, pivaloyl(Piv), tetrahydropyranyl (THP), tetrahydrofuranyl (THF), trityl (Trt),triisopropylsilyl (TIPS), tert-butyldiphenylsilyl (TBDPS), and acetate;and

is an optional double bond.

In one aspect, there is provided a compound of Formula (1):

L-(N)_(n)   (1)

wherein L is selected from an ethylene glycol chain, an alkyl chain, apeptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, anester, an amide, a triazole, and combinations thereof, wherein Formula(1) optionally further comprises one or more branch point Bp, and one ormore spacer S, wherein Bp is independently for each occurrence apolyvalent organic species or derivative thereof; S is independently foreach occurrence selected from an ethylene glycol chain, an alkyl chain,a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, anester, an amide, a triazole, and combinations thereof; N is an RNAduplex comprising a sense strand and an antisense strand, wherein thesense strand and antisense strand each independently comprise one ormore chemical modifications; and n is 2, 3, 4, 5, 6, 7 or 8, whereinat least one N includes a modified intersubunit linkage of Formula (I):

wherein:

each B is, independently, a base pairing moiety;

W is selected from the group consisting of O, OCH₂, OCH, CH₂, and CH,optionally wherein W is selected from the group consisting of OCH₂ andOCH;

each X is, independently, selected from the group consisting of halo(e.g., fluoro or chloro), hydroxy, and C₁₋₆ alkoxy optionally whereineach X is, independently, selected from the group consisting of halo andC₁₋₆ alkoxy (e.g., methoxy, ethoxy, n-propoxy, sec-propoxy, n-butoxy,sec-butoxy, tert-butoxy, n-pentoxy, or n-heptoxy);

Y is selected from the group consisting of O⁻, OH, OR, NH⁻, NH₂, S⁻, andSH, optionally wherein Y is selected from the group consisting of O⁻,OH, and OR;

Z is selected from the group consisting of O and CH₂;

R is a protecting group; and

-   -   is an optional double bond.

In some embodiments, W is OCH₂.

In some embodiments, W is OCH and

is a double bond.

In some embodiments, Z is O.

In some embodiments, Z is CH₂

In some embodiments, the compound has a structure selected from formulas(1-1)-(1-9):

In some embodiments, the antisense strand comprises a 5′ terminal groupR selected from the group consisting of:

In some embodiments, the compound has the structure of Formula (2):

wherein

X, for each occurrence, independently, is selected from adenosine,guanosine, uridine, cytidine, and chemically-modified derivativesthereof;

Y, for each occurrence, independently, is selected from adenosine,guanosine, uridine, cytidine, and chemically-modified derivativesthereof;

- represents a phosphodiester internucleoside linkage;

= represents a phosphorothioate internucleoside linkage; and

--- represents, individually for each occurrence, a base-pairinginteraction or a mismatch,

with the proviso that at least one of the - linkages or at least one ofthe = linkages of Formula (2) be a modified subunit linkage of Formula(I).

In some embodiments, the compound has the structure of Formula (3):

wherein

X, for each occurrence, independently, is a nucleotide comprising a2′-deoxy-2′-fluoro modification;

X, for each occurrence, independently, is a nucleotide comprising a2′-O-methyl modification;

Y, for each occurrence, independently, is a nucleotide comprising a2′-deoxy-2′-fluoro modification; and

Y, for each occurrence, independently, is a nucleotide comprising a2′-O-methyl modification, and

with the proviso that at least one of the - linkages or at least one ofthe = linkages of Formula (3) be a modified subunit linkage of Formula(I).

In some embodiments, the compound has the structure of Formula (4):

whereinX, for each occurrence, independently, is selected from adenosine,guanosine, uridine, cytidine, and chemically-modified derivativesthereof;Y, for each occurrence, independently, is selected from adenosine,guanosine, uridine, cytidine, and chemically-modified derivativesthereof;

- represents a phosphodiester internucleoside linkage;

= represents a phosphorothioate internucleoside linkage; and

--- represents, individually for each occurrence, a base-pairinginteraction or a mismatch,

with the proviso that at least one of the - linkages or at least one ofthe = linkages of Formula (4) be a modified subunit linkage of Formula(I).

In some embodiments, the compound has a structure of Formula (5):

wherein

X, for each occurrence, independently, is a nucleotide comprising a2′-deoxy-2′-fluoro modification;

X, for each occurrence, independently, is a nucleotide comprising a2′-O-methyl modification;

Y, for each occurrence, independently, is a nucleotide comprising a2′-deoxy-2′-fluoro modification; and

Y, for each occurrence, independently, is a nucleotide comprising a2′-O-methyl modification.

In some embodiments, moiety L has the structure of L1:

Moiety R may be R³ and n may be 2.

In some embodiments, L has the structure of L2:

Moiety R may be R3 and n may be 2.

In some embodiments, when Y is O⁻, either Z or W is not O.

In some embodiments, Z is CH2 and W is CH2.

In some embodiments, the modified intersubunit linkage of Formula (I) isa modified intersubunit linkage of Formula (II):

In some embodiments, Z is CH2 and W is O.

In some embodiments, the modified intersubunit linkage of Formula (I) isa modified intersubunit linkage of Formula (III):

In some embodiments, Z is O and W is CH₂.

In some embodiments, the modified intersubunit linkage of Formula (I) isa modified intersubunit linkage of Formula (IV):

In some embodiments, Z is O and W is CH.

In some embodiments, the modified intersubunit linkage of Formula (I) isa modified intersubunit linkage of Formula (V):

In some embodiments, Z is O and W is OCH2.

In some embodiments, the modified intersubunit linkage of Formula I is amodified intersubunit linkage of Formula VI:

In some embodiments of Formula VI:

-   -   each B is, independently, a base pairing moiety;    -   each X is, independently, selected from the group consisting of        halo, hydroxy, and C₁₋₆ alkoxy; optionally wherein each X is,        independently, selected from the group consisting of halo (e.g.,        fluoro) and C₁₋₆ alkoxy (e.g., methoxy, ethoxy, n-propoxy,        sec-propoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, or        n-heptoxy);    -   Y is selected from the group consisting of O⁻, OH, OR, NH⁻, NH₂,        S⁻, and SH, optionally wherein Y is selected from the group        consisting of O⁻, OH, and OR;    -   Z is selected from the group consisting of O and CH₂; and    -   is an optional double bond.

In some embodiments of Formula VI:

-   -   each X is, independently, selected from the group consisting of        fluoro, hydroxy, and C₁₋₆ alkoxy; optionally wherein each X is,        independently, selected from the group consisting of fluoro and        C₁₋₆ alkoxy (e.g., methoxy, ethoxy, n-propoxy, sec-propoxy,        n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, or n-heptoxy);    -   Y is selected from the group consisting of O⁻, OH, and OR;    -   Z is selected from the group consisting of O and CH₂; and    -   is an optional double bond.

In some embodiments of Formula VI:

-   -   each X is, independently, selected from the group consisting of        fluoro, hydroxy, methoxy, ethoxy, n-propoxy, sec-propoxy,        n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, and n-heptoxy;    -   Y is selected from the group consisting of O⁻, OH, and OR;    -   Z is selected from the group consisting of O and CH₂; and    -   is an optional double bond.

In some embodiments of Formula VI:

-   -   each X is, independently, selected from the group consisting of        fluoro, hydroxy, methoxy, ethoxy, n-propoxy, sec-propoxy,        n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, and n-heptoxy;    -   Y is selected from the group consisting of O⁻, OH, and OR;    -   Z is O; and    -   is an optional double bond.

In some embodiments of Formula VI:

-   -   each X is, independently, selected from the group consisting of        fluoro, hydroxy, methoxy, ethoxy, n-propoxy, sec-propoxy,        n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, and n-heptoxy;    -   Y is selected from the group consisting of O⁻, OH, and OR;    -   Z is CH₂; and    -   is an optional double bond.

In some embodiments of Formula VI:

-   -   each X is, independently, selected from the group consisting of        fluoro, hydroxy, and methoxy;    -   Y is selected from the group consisting of O⁻, OH, and OR;    -   Z is O; and    -   is an optional double bond.

In some embodiments of Formula VI:

-   -   each X is, independently, selected from the group consisting of        fluoro, hydroxy, and methoxy;    -   Y is selected from the group consisting of O⁻, OH, and OR;    -   Z is CH₂; and    -   is an optional double bond.

In some embodiments, the modified intersubunit linkage of Formula (I) isa modified intersubunit linkage of Formula (VIa):

In some embodiments, Z is CH2 and W is CH.

In some embodiments, the modified intersubunit linkage of Formula (I) isa modified intersubunit linkage of Formula (VII):

In some embodiments, the base pairing moiety B is selected from thegroup consisting of adenine, guanine, cytosine, and uracil.

In one aspect, the disclosure provides a delivery system for therapeuticnucleic acids having the structure of Formula (6):

L-(cNA)_(n)   (6)

wherein L is selected from an ethylene glycol chain, an alkyl chain, apeptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, anester, an amide, a triazole, and combinations thereof, wherein formula(6) optionally further comprises one or more branch point Bp, and one ormore spacer S, wherein Bp is independently for each occurrence apolyvalent organic species or derivative thereof; S is independently foreach occurrence selected from an ethylene glycol chain, an alkyl chain,a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, anester, an amide, a triazole, and combinations thereof; each cNA,independently, is a carrier nucleic acid comprising one or more chemicalmodifications; and n is 2, 3, 4, 5, 6, 7 or 8,wherein at least one chemical modification of at least one cNA is anintersubunit linkage of Formula (I):

wherein:

each B is, independently, a base pairing moiety;

W is selected from the group consisting of O, OCH₂, OCH, CH₂, and CH,optionally wherein W is selected from the group consisting of OCH₂ andOCH;

each X is, independently, selected from the group consisting of halo(e.g., fluoro or chloro), hydroxy, and C₁₋₆ alkoxy, optionally whereineach X is, independently, selected from the group consisting of halo andC₁₋₆ alkoxy (e.g., methoxy, ethoxy, n-propoxy, sec-propoxy, n-butoxy,sec-butoxy, tert-butoxy, n-pentoxy, or n-heptoxy);

Y is selected from the group consisting of O⁻, OH, OR, NH⁻, NH₂, S, andSH, optionally wherein Y is selected from the group consisting of O⁻,OH, and OR.

Z is selected from the group consisting of O and CH₂;

R is a protecting group; and

is an optional double bond.

In some embodiments, W is OCH₂.

In some embodiments, W is OCH and

is a double bond.

In some embodiments, Z is O.

In some embodiments, Z is CH₂

In some embodiments, the delivery system has a structure selected fromformulas (6-1)-(6-9):

In some embodiments, each cNA independently comprises at least 15contiguous nucleotides.

In some embodiments, each cNA independently consists ofchemically-modified nucleotides.

In some embodiments, the delivery system further comprises n therapeuticnucleic acids (NA), wherein each NA is hybridized to at least one cNA.

In some embodiments, each NA independently comprises at least 16contiguous nucleotides.

In some embodiments, each NA independently comprises 16-20 contiguousnucleotides.

In some embodiments, each NA comprises an unpaired overhang of at least2 nucleotides.

In some embodiments, the nucleotides of the overhang are connected viaphosphorothioate linkages.

In some embodiments, each NA, independently, is selected from the groupconsisting of: DNA, siRNAs, antagomiRs, miRNAs, gapmers, mixmers, orguide RNAs.

In some embodiments, wherein each NA is the same.

In some embodiments, each NA is not the same.

In some embodiments, the target of delivery is selected from the groupconsisting of: brain, liver, skin, kidney, spleen, pancreas, colon, fat,lung, muscle, and thymus.

In some embodiments, when Y is O—, either Z or W is not O.

In some embodiments, Z is CH₂ and W is CH₂.

In some embodiments, the modified intersubunit linkage of Formula (I) isa modified intersubunit linkage of Formula (II):

In some embodiments, Z is CH₂ and W is O.

In some embodiments, the modified intersubunit linkage of Formula (I) isa modified intersubunit linkage of Formula (III):

In some embodiments, Z is O and W is CH₂.

In some embodiments, the modified intersubunit linkage of Formula (I) isa modified intersubunit linkage of Formula (IV):

In some embodiments, Z is O and W is CH.

In some embodiments, the modified intersubunit linkage of Formula (I) isa modified intersubunit linkage of Formula (V):

In some embodiments, Z is O and W is OCH₂.

In some embodiments, the modified intersubunit linkage of Formula I is amodified intersubunit linkage of Formula VI:

In some embodiments of Formula VI:

-   -   each B is, independently, a base pairing moiety;    -   each X is, independently, selected from the group consisting of        halo, hydroxy, and C₁₋₆ alkoxy; optionally wherein each X is,        independently, selected from the group consisting of halo (e.g.,        fluoro) and C₁₋₆ alkoxy (e.g., methoxy, ethoxy, n-propoxy,        sec-propoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, or        n-heptoxy);    -   Y is selected from the group consisting of O⁻, OH, OR, NH⁻, NH₂,        S⁻, and SH, optionally wherein Y is selected from the group        consisting of O⁻, OH, and OR;    -   Z is selected from the group consisting of O and CH₂; and    -   is an optional double bond.

In some embodiments of Formula VI:

-   -   each X is, independently, selected from the group consisting of        fluoro, hydroxy, and C₁₋₆ alkoxy; optionally wherein each X is,        independently, selected from the group consisting of fluoro and        C₁₋₆ alkoxy (e.g., methoxy, ethoxy, n-propoxy, sec-propoxy,        n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, or n-heptoxy);    -   Y is selected from the group consisting of O⁻, OH, and OR;    -   Z is selected from the group consisting of O and CH₂; and    -   is an optional double bond.

In some embodiments of Formula VI:

-   -   each X is, independently, selected from the group consisting of        fluoro, hydroxy, methoxy, ethoxy, n-propoxy, sec-propoxy,        n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, and n-heptoxy;    -   Y is selected from the group consisting of O⁻, OH, and OR;    -   Z is selected from the group consisting of O and CH₂; and    -   is an optional double bond.

In some embodiments of Formula VI:

-   -   each X is, independently, selected from the group consisting of        fluoro, hydroxy, methoxy, ethoxy, n-propoxy, sec-propoxy,        n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, and n-heptoxy;    -   Y is selected from the group consisting of O⁻, OH, and OR;    -   Z is O; and    -   is an optional double bond.

In some embodiments of Formula VI:

-   -   each X is, independently, selected from the group consisting of        fluoro, hydroxy, methoxy, ethoxy, n-propoxy, sec-propoxy,        n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, and n-heptoxy;    -   Y is selected from the group consisting of O⁻, OH, and OR;    -   Z is CH₂; and    -   is an optional double bond.

In some embodiments of Formula VI:

-   -   each X is, independently, selected from the group consisting of        fluoro, hydroxy, and methoxy;    -   Y is selected from the group consisting of O⁻, OH, and OR;    -   Z is O; and    -   is an optional double bond.

In some embodiments of Formula VI:

-   -   each X is, independently, selected from the group consisting of        fluoro, hydroxy, and methoxy;    -   Y is selected from the group consisting of O⁻, OH, and OR;    -   Z is CH₂; and    -   is an optional double bond.

In some embodiments, the modified intersubunit linkage of Formula (I) isa modified intersubunit linkage of Formula (VIa):

In some embodiments, Z is CH₂ and W is CH.

In some embodiments, the modified intersubunit linkage of Formula (I) isa modified intersubunit linkage of Formula (VII):

In some embodiments, the base pairing moiety B is selected from thegroup consisting of adenine, guanine, cytosine, and uracil.

In some embodiments of any of the preceding aspects of the disclosure,the RNA molecule (e.g., siRNA) is from 8 nucleotides to 80 nucleotidesin length (e.g., 8 nucleotides, 9 nucleotides, 10 nucleotides, 11nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27nucleotides, 28 nucleotides, 29 nucleotides, 30 nucleotides, 31nucleotides, 32 nucleotides, 33 nucleotides, 34 nucleotides, 35nucleotides, 36 nucleotides, 37 nucleotides, 38 nucleotides, 39nucleotides, 40 nucleotides, 41 nucleotides, 42 nucleotides, 43nucleotides, 44 nucleotides, 45 nucleotides, 46 nucleotides, 47nucleotides, 48 nucleotides, 49 nucleotides, 50 nucleotides, 51nucleotides, 52 nucleotides, 53 nucleotides, 54 nucleotides, 55nucleotides, 56 nucleotides, 57 nucleotides, 58 nucleotides, 59nucleotides, 60 nucleotides, 61 nucleotides, 62 nucleotides, 63nucleotides, 64 nucleotides, 65 nucleotides, 66 nucleotides, 67nucleotides, 68 nucleotides, 69 nucleotides, 70 nucleotides, 71nucleotides, 72 nucleotides, 73 nucleotides, 74 nucleotides, 75nucleotides, 76 nucleotides, 77 nucleotides, 78 nucleotides, 79nucleotides, or 80 nucleotides in length).

In some embodiments, the RNA molecule is from 10 to 50 nucleotides inlength (e.g., 10 nucleotides, 11 nucleotides, 12 nucleotides, 13nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29nucleotides, 30 nucleotides, 31 nucleotides, 32 nucleotides, 33nucleotides, 34 nucleotides, 35 nucleotides, 36 nucleotides, 37nucleotides, 38 nucleotides, 39 nucleotides, 40 nucleotides, 41nucleotides, 42 nucleotides, 43 nucleotides, 44 nucleotides, 45nucleotides, 46 nucleotides, 47 nucleotides, 48 nucleotides, 49nucleotides, or 50 nucleotides in length).

In some embodiments, the RNA molecule comprises about 15 nucleotides toabout 25 nucleotides in length. In some embodiments, the RNA molecule isfrom 15 to 25 nucleotides in length (e.g., 15 nucleotides, 16nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24nucleotides, or 25 nucleotides in length).

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the present inventionwill be more fully understood from the following detailed description ofillustrative embodiments taken in conjunction with the accompanyingdrawings. The patent or application file contains at least one drawingexecuted in color. Copies of this patent or patent applicationpublication with color drawing(s) will be provided by the Office uponrequest and payment of the necessary fee.

FIG. 1 summarizes the modified intersubunit linkers provided herein.

FIG. 2A shows a representative example for preparing a monomer for themodified phosphinate-containing oligonucleotides provided herein.

FIG. 2B shows a representative example for preparing another monomer forthe modified phosphinate-containing oligonucleotides provided herein.

FIG. 2C shows representative example for preparing a modifiedphosphinate-containing oligonucleotide provided herein.

FIG. 3A shows a representative example for preparing a monomer for themodified phosphonate-containing oligonucleotides provided herein.

FIG. 3B shows representative example for preparing a modifiedphosphonate-containing oligonucleotide provided herein.

FIG. 4 provides a representative method for preparing theoligonucleotides provided herein on solid support.

FIG. 5 shows a method for preparing a vinylphosphinate-modifiedoligonucleotide.

FIG. 6 shows a representative example for preparing a monomer for thesynthesis of the oligonucleotides provided herein.

FIG. 7 shows a representative example for preparing a monomer for thesynthesis of the modified oligonucleotides provided herein.

FIG. 8 illustrates the method for preparing the modifiedoligonucleotides provided herein.

FIG. 9A illustrates the impact of internal-VP modification on siRNAthermal stability.

FIG. 9B illustrates the impact of internal-VP modification on duplex RNAthermal stability.

FIG. 10A shows RNA stability in a digestion test by SVPD (3′'5′exonuclease) ×4 condition for RNA having PO, VP, and PS linkers.

FIG. 10B shows RNA stability in a digestion test by SVPDE (3′→5′exonuclease) ×10 condition for RNA having PO, VP, and PS linkers.

FIG. 10C shows RNA stability in a digestion test by SVPDE (3′→5′exonuclease) for VP/PS mixed sequences.

FIG. 11 illustrates the effect of adding a mismatch in the siRNAsequence to improve allelic discrimination without impairing silencingof the mutant allele.

FIG. 12 illustrates the silencing efficacy of VP-modified siRNA.

FIG. 13 depicts a method for preparing oligonucleotides having a vinylphosphonate modified intersubunit linkage described herein.

FIG. 14 illustrates the sequences of vinyl phosphonate modifiedoligonucleotides synthesized. Antisense strands are depicted 5′ to 3′,with the SNP site in red and the mismatch in blue.

FIG. 15 is a schematic representation of hsiRNA antisense scaffoldsaligned to the HTT sequence surrounding SNP site rs362273, wherein thegreen box indicates the position of the SNP site.

FIG. 16 depicts the change in mRNA expression when standard siRNA isused versus VP-modified siRNA.

FIG. 17 shows on or off-target HTT-mRNA knock down with control andVP-modified siRNA.

FIG. 18 shows the structure of Di-hsiRNAs. Black—2′-O-methyl,grey—2′-fluoro, red dash—phosphorothioate bond, linker—tetraethyleneglycol. Di-hsiRNAs are two asymmetric siRNAs attached through the linkerat the 3′ ends of the sense strand. Hybridization to the longerantisense strand creates protruding single stranded fullyphosphorthioated regions, essential for tissue distribution, cellularuptake and efficacy. The structures presented utilize teg linger of fourmonomers. The chemical identity of the linker can be modified withoutthe impact on efficacy. It can be adjusted by length, chemicalcomposition (fully carbon), saturation or the addition of chemicaltargeting ligands.

FIG. 19 shows a chemical synthesis, purification and quality control ofDi-branched siRNAs.

FIG. 20 shows HPLC and quality control of compounds produced by themethod depicted in FIG. 19. Three major products were identified by massspectrometry as sense strand with TEG (tetraethylene glycol) linker,di-branched oligo and Vit-D (calciferol) conjugate. All products wherepurified by HPLC and tested in vivo independently. Only Di-branchedoligo is characterized by unprecedented tissue distribution andefficacy, indicating that branching structure is essential for tissueretention and distribution.

FIG. 21 shows mass spectrometry confirming the mass of the Di-branchedoligonucleotide. The observed mass of 11683 corresponds to two sensestrands attached through the TEG linker by the 3′ ends.

FIG. 22 shows a synthesis of a branched oligonucleotide usingalternative chemical routes.

FIG. 23 shows exemplary amidite linkers, spacers and branching moieties.

FIG. 24 shows oligonucleotide branching motifs. The double-helicesrepresented oligonucleotides. The combination of different linkers,spacer and branching points allows generation of a wide diversity ofbranched hsiRNA structures.

FIG. 25 shows structurally diverse branched oligonucleotides.

FIG. 26 shows an asymmetric compound of the invention having foursingle-stranded phosphorothioate regions.

FIGS. 27A-27C show in vitro efficacy data. (FIG. 27A) HeLa cells weretransfected (using RNAiMax) with Di-branched oligo at concentrationsshown for 72 hours. (FIG. 27B) Primary cortical mouse neurons weretreated with Di-branched oligo at concentrations shown for 1 week. mRNAwas measured using Affymetrix Quantigene 2.0. Data was normalized tohousekeeping gene (PPIB) and graphed as % of untreated control. (FIG.27C) HeLa cells were treated passively (no formulation) with Di-siRNAoligo at concentrations shown for 1 week.

FIGS. 28A-28B show brain distribution of Di-siRNA or TEG only after 48hours following intra-striatal injection. Intrastriatal injection of 2nmols of (FIG. 28A) Di-branched oligo (4 nmols of correspondingantisense strand) or (FIG. 28B) TEG-oligo only. N=2 mice per conjugate.Brains collected 48 hours later and stained with Dapi (nuclei, blue).Red-oligo. The left side of brain in (FIG. 28A) appears bright red,whereas the left side of the brain in (FIG. 28B) only faintly red.

FIG. 29 shows that a single injection of Di-siRNA was detected bothipsilateral and contralateral to the injection site.

FIGS. 30A-30B shows Di-hsiRNA wide distribution and efficacy in mousebrain. (FIG. 30A) Robust Htt mRNA silencing in both Cortex and Striatum7 days after single IS injection (25 μg), QuantiGene®. (FIG. 30B) Levelsof hsiRNA accumulation in tissues 7 days after injection (PNA assay).

FIGS. 31A-31C show wide distribution and efficacy throughout the spinalcord following bolus intrathecal injection of Di-hsiRNA. Intrathecalinjection in lumbar of 3 nmols Di-branched Oligo (6 nmols ofcorresponding antisense HTT strand). (FIG. 31A) Robust Htt mRNAsilencing in all region of spinal cord, 7 days, n-6. Animals sacrificed7 days post-injection. Tissue punches taken from cervical, thoracic andlumbar regions of spinal cord. mRNA was quantified using AffymetrixQuantigene 2.0 as per Coles et al. 2015. Data normalized to housekeepinggene, HPRT, and graft as percent of aCSF control. aCSF—artificial CSF.(FIG. 31B) Animals were injected lumbar IT with 75 μg ofCy3-Chol-hsiRNA, Cy-Di-hsiRNA. Chol-hsiRNAs shows steep gradient ofdiffusion from outside to inside of spinal cord. Di-hsiRNAs shows widedistribution throughout the cord (all regions). Leica 10× (20 mm bar).Image of Di-branched oligo in cervical region of spinal cord 48 hoursafter intrathecal injection. Red=oligo, Blue=Dapi. (FIG. 31C) Image ofDi-branched oligo in liver 48 hours after intrathecal injection.Red=oligo, Blue=Dapi.

FIGS. 32A-32C show branched oligonucleotides of the invention, (FIG.32A) formed by annealing three oligonucleotides. The longer linkingoligonucleotides may comprise a cleavable region in the form ofunmodified RNA, DNA or UNA; (FIG. 32B) asymmetrical branchedoligonucleotides with 3′ and 5′ linkages to the linkers or spacesdescribed previously. This can be applied the 3′ and 5′ ends of thesense strand or the antisense strands or a combination thereof, (FIG.32C) branched oligonucleotides made up of three separate strands. Thelong dual sense strand can be synthesized with 3′ phosphoramidites and5′ phosphoramidites to allow for 3′-3′ adjacent or 5′-5′ adjacent ends.

FIG. 33 shows branched oligonucleotides of the invention with conjugatedbioactive moieties.

FIG. 34 shows the relationship between phosphorothioate content andstereoselectivity.

FIG. 35 depicts exemplary hydrophobic moieties.

FIG. 36 depicts exemplary internucleotide linkages.

FIG. 37 depicts exemplary internucleotide backbone linkages.

FIG. 38 depicts exemplary sugar modifications.

FIGS. 39A-39C depict Di-FM-hsiRNA. (FIG. 39A) Chemical composition ofthe four sub-products created from VitD-FM-hsiRNA synthesis and crudereverse phase analytical HPLC of the original chemical synthesis. (FIG.39B) Efficacy of sub-products in HeLa cells after lipid mediateddelivery of hsiRNA. Cells were treated for 72 hours. mRNA was measuredusing QuantiGene 2.0 kit (Affymetrix). Data are normalized tohousekeeping gene HPRT and presented as a percent of untreated control.(FIG. 39C) A single, unilateral intrastriatal injection (25 μg) of eachhsiRNA sub-product. Images taken 48 hours after injection.

FIGS. 40A-40B show that Di-HTT-Cy3 does not effectively induce silencingin the liver or kidneys following intrastriatal injection. FIG. 40Adepicts a scatter dot plot showing Htt mRNA expression in the liverone-week post intrastriatal injection of Di-HTT-Cy3 compared to anegative control (aCSF). FIG. 40B depicts a scatter dot plot showing HttmRNA expression in the kidney one-week post intrastriatal injection ofDi-HTT-Cy3 compared to a negative control (aCSF).

FIGS. 41A-41B shows that Di-HTT effectively silences HTT gene expressionin both the striatum and the cortex following intrastriatal injectionand that Di-HTT-Cy3 is slightly more efficacious than Di-HTT(unlabeled). FIG. 41A depicts a scatter dot plot showing Htt mRNAexpression in the striatum one-week post intrastriatal injection ofDi-HTT, Di-HTT-Cy3, or two negative controls (aCSF or Di-NTC). FIG. 41Bdepicts a scatter dot plot showing Htt mRNA expression in the cortexone-week post intrastriatal injection of Di-HTT, Di-HTT-Cy3, or twonegative controls (aCSF or Di-NTC).

FIG. 42 depicts a scatter dot plot measuring Di-HTT-Cy3 levels in thestriatum and cortex. The plot shows that significant levels ofDi-HTT-Cy3 are still detectable two weeks post intrastriatal injection.

FIGS. 43A-43B show that Di-HTT-Cy3 effectively silences HTT mRNA andprotein expression in both the striatum and the cortex two weeks postintrastriatal injection. FIG. 43A depicts a scatter dot plot measuringHtt mRNA levels in the striatum and cortex two weeks post injection.FIG. 43B depicts a scatter dot plot measuring Htt protein levels in thestriatum and cortex two weeks post injection.

FIGS. 44A-44B show that high dose Di-HTT-Cy3 treatment does not causesignificant toxicity in vivo but does lead to significant gliosis invivo two weeks post intrastriatal injection. FIG. 44A depicts a scatterdot plot measuring DARPP32 signal in the striatum and cortex two weeksafter injection with Di-HTT-Cy3 or aCSF. FIG. 44B depicts a scatter dotplot measuring GFAP protein levels in the striatum and cortex two weeksafter injection with Di-HTT-Cy3 or aCSF.

FIG. 45 depicts fluorescent imaging showing that intrathecal injectionof Di-HTT-Cy3 results in robust and even distribution throughout thespinal cord.

FIG. 46 depicts a merged fluorescent image of FIG. 45 (zoom of spinalcord). Blue-nuclei, red-Di-HTT-Cy3.

FIGS. 47A-47C shows the widespread distribution of Di-HTT-Cy3 48 hourspost intracerebroventricular injection. FIG. 47A depicts fluorescentimaging of sections of the striatum, cortex, and cerebellum. FIG. 47Bdepicts brightfield images of the whole brain injected with control(aCSF) or Di-HTT-Cy3. FIG. 47C depicts a fluorescent image of a wholebrain section 48 hours after Di-HTT-Cy3 injection.

FIG. 48 shows that Di-HTT-Cy3 accumulates in multiple brain regions twoweeks post intracerebroventricular injection. A scatter dot plotmeasures the level of Di-HTT-Cy3 in multiple areas of the brain.

FIG. 49A shows that Di-HTT-Cy3 induces Htt gene silencing in multipleregions of the brain two weeks post intracerebroventricular injectioncompared to a negative control injection (aCSF). A scatter dot plotmeasures Htt mRNA levels in multiple areas of the brain. FIG. 49B showsthat Di-HTT-Cy3 induces Htt silencing in multiple regions of the braintwo weeks post intracerebroventricular injection compared to a negativecontrol injection (aCSF). A scatter dot plot measures Htt protein levelsin multiple areas of the brain.

FIG. 50 shows that intracerebroventricular injection of high doseDi-HTT-Cy3 causes minor toxicity in vivo. A scatter dot plot measuresDARPP32 signal in multiple regions of the brain following Di-HTT-Cy3 ofaCSF injection.

FIG. 51 shows that intracerebroventricular injection of high doseDi-HTT-Cy3 causes significant gliosis in vivo. A scatter dot plotmeasures DARPP32 signal in multiple regions of the brain followingDi-HTT-Cy3 of aCSF injection.

FIG. 52 shows that Di-HTT-Cy3 is distributed to multiple organsfollowing intravenous injection. Fluorescent images depict Di-HTT-Cy3levels in the heart, kidney, adrenal gland, and spleen followingintravenous injection of Di-HTT-Cy3 or a negative control (PBS).

FIG. 53 shows that Di-HTT-Cy3 accumulates in multiple organs followingintravenous injection. A scatter dot plot measures the levels ofDi-HTT-Cy3 in multiple tissues.

FIG. 54 illustrates the structures of hsiRNA and fully metabolized (FM)hsiRNA.

FIGS. 55A-55B show that full metabolic stabilization of hsiRNAs resultsin more efficacious gene silencing following intrastriatal injection ofhsiRNA HTT or FM-hsiRNA HTT. FIG. 55A depicts a scatter dot plotmeasuring HTT mRNA levels up to 12 days after intrastriatal injection.FIG. 55B depicts a scatter dot plot measuring HTT mRNA levels up to 28days after intrastriatal injection.

FIG. 56 depicts the chemical diversity of single stranded fully modifiedoligonucleotides. The single stranded oligonucleotides can consist ofgapmers, mixmers, miRNA inhibitors, SSOs, PMOs, or PNAs.

FIG. 57 depicts Di-HTT with a TEG phosphoramidate linker.

FIG. 58 depicts Di-HTT with a TEG di-phosphate linker.

FIG. 59 depicts variations of Di-HTT with either two oligonucleotidebranches or four oligonucleotide branches.

FIG. 60 depicts another variant of Di-HTT of a structure with twooligonucleotide branches and R2 attached to the linker.

FIG. 61 depicts a first strategy for the incorporation of a hydrophobicmoiety into the branched oligonucleotide structures.

FIG. 62 depicts a second strategy for the incorporation of a hydrophobicmoiety into the branched oligonucleotide structures.

FIG. 63 depicts a third strategy for the incorporation of a hydrophobicmoiety into the branched oligonucleotide structures.

FIG. 64 depicts in vitro silencing efficacy of target mRNA with siRNAduplexes containing ex-NA intersubunit linkages are various positions.

FIG. 65 depicts the ex-NA (2′O-Methyl) phosphoramidite synthesis scheme.

FIG. 66 depicts the ex-NA (2′-Fluoro) phosphoramidite synthesis scheme.

FIG. 67 depicts the coupling of ex-NA phosphoramidite on solid support.

DETAILED DESCRIPTION OF CERTAIN EXEMPLARY EMBODIMENTS

Novel siRNAs are provided. Also provided are novel oligonucleotides.

Unless otherwise specified, nomenclature used in connection with celland tissue culture, molecular biology, immunology, microbiology,genetics and protein and nucleic acid chemistry and hybridizationdescribed herein are those well-known and commonly used in the art.Unless otherwise specified, the methods and techniques provided hereinare performed according to conventional methods well known in the artand as described in various general and more specific references thatare cited and discussed throughout the present specification unlessotherwise indicated. Enzymatic reactions and purification techniques areperformed according to manufacturer's specifications, as commonlyaccomplished in the art or as described herein. The nomenclature used inconnection with, and the laboratory procedures and techniques of,analytical chemistry, synthetic organic chemistry, and medicinal andpharmaceutical chemistry described herein are those well-known andcommonly used in the art. Standard techniques are used for chemicalsyntheses, chemical analyses, pharmaceutical preparation, formulation,and delivery, and treatment of patients.

Unless otherwise defined herein, scientific and technical terms usedherein have the meanings that are commonly understood by those ofordinary skill in the art. In the event of any latent ambiguity,definitions provided herein take precedent over any dictionary orextrinsic definition. Unless otherwise required by context, singularterms shall include pluralities and plural terms shall include thesingular. The use of “or” means “and/or” unless stated otherwise. Theuse of the term “including,” as well as other forms, such as “includes”and “included,” is not limiting.

So that the invention may be more readily understood, certain terms arefirst defined.

The term “nucleoside” refers to a molecule having a purine or pyrimidinebase covalently linked to a ribose or deoxyribose sugar. Exemplarynucleosides include adenosine, guanosine, cytidine, uridine andthymidine. Additional exemplary nucleosides include inosine, 1-methylinosine, pseudouridine, 5,6-dihydrouridine, ribothymidine,2N-methylguanosine and 2,2N,N-dimethylguanosine (also referred to as“rare” nucleosides). The term “nucleotide” refers to a nucleoside havingone or more phosphate groups joined in ester linkages to the sugarmoiety. Exemplary nucleotides include nucleoside monophosphates,diphosphates and triphosphates. The terms “polynucleotide” and “nucleicacid molecule” are used interchangeably herein and refer to a polymer ofnucleotides joined together by a phosphodiester or phosphorothioatelinkage between 5′ and 3′ carbon atoms.

As used herein, “gene editing complex” refers to a biologically activemolecule (e.g., a protein, one or more proteins, a nucleic acid, one ormore nucleic acids, or any combination of the foregoing) configured foradding, disrupting or changing genomic sequences (e.g., a gene sequence)by causing a genetic lesion (e.g., double stranded break (DSB) or singlestranded break (SSB)) in a target DNA or other target nucleic acid,which may be loaded into an artificial exosome of the disclosure ascargo. The genetic lesion may be introduced in a number of ways known inthe art. Examples of gene editing complexes include but are not limitednucleases such as transcription activator-like effector nucleases(TALENs), zinc finger nucleases (ZFNs), engineered meganucleasere-engineered homing endonucleases, the CRISPR/Cas system, andmeganucleases (e.g., Meganuclease I-Scel). In some embodiments, a geneediting complex comprises proteins or molecules (e.g., components)related to the CRISPR system, including but not limited to Cas9, Cas6,dCas9, CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA), andvariants thereof. In some embodiments, the Cas protein is a Cpflprotein, or a variant thereof.

As used herein, the terms “endonuclease” and “nuclease” refer to anenzyme that cleaves a phosphodiester bond or bonds within apolynucleotide chain. Nucleases may be naturally occurring orgenetically engineered. Genetically engineered nucleases areparticularly useful for genome editing and are generally classified intofour families: zinc finger nucleases (ZFNs), transcriptionactivator-like effector nucleases (TALENs), meganucleases (e.g.,engineered meganucleases) and RNA guides nucleases such as theCRISPR-associated proteins (Cas nucleases).

The term “RNA” or “RNA molecule” or “ribonucleic acid molecule” refersto a polymer of ribonucleotides (e.g., 2, 3, 4, 5, 10, 15, 20, 25, 30,or more ribonucleotides). The term “DNA” or “DNA molecule” or“deoxyribonucleic acid molecule” refers to a polymer ofdeoxyribonucleotides. DNA and RNA can be synthesized naturally (e.g., byDNA replication or transcription of DNA, respectively). RNA can bepost-transcriptionally modified. DNA and RNA can also be chemicallysynthesized. DNA and RNA can be single-stranded (i.e., ssRNA and ssDNA,respectively) or multi-stranded (e.g., double stranded, i.e., dsRNA anddsDNA, respectively). “mRNA” or “messenger RNA” is single-stranded RNAthat specifies the amino acid sequence of one or more polypeptidechains. This information is translated during protein synthesis whenribosomes bind to the mRNA.

As used herein, the term “small interfering RNA” (“siRNA”) (alsoreferred to in the art as “short interfering RNAs”) refers to an RNA (orRNA analog) comprising between about 10-50 nucleotides (or nucleotideanalogs) which is capable of directing or mediating RNA interference.Preferably, a siRNA comprises between about 15-30 nucleotides ornucleotide analogs, more preferably between about 16-25 nucleotides (ornucleotide analogs), even more preferably between about 18-23nucleotides (or nucleotide analogs), and even more preferably betweenabout 19-22 nucleotides (or nucleotide analogs) (e.g., 19, 20, 21 or 22nucleotides or nucleotide analogs). The term “short” siRNA refers to asiRNA comprising about 21 nucleotides (or nucleotide analogs), forexample, 19, 20, 21 or 22 nucleotides. The term “long” siRNA refers to asiRNA comprising about 24-25 nucleotides, for example, 23, 24, 25 or 26nucleotides. Short siRNAs may, in some instances, include fewer than 19nucleotides, e.g., 16, 17 or 18 nucleotides, provided that the shortersiRNA retains the ability to mediate RNAi. Likewise, long siRNAs may, insome instances, include more than 26 nucleotides, provided that thelonger siRNA retains the ability to mediate RNAi absent furtherprocessing, e.g., enzymatic processing, to a short siRNA.

The term “nucleotide analog” or “altered nucleotide” or “modifiednucleotide” refers to a non-standard nucleotide, including non-naturallyoccurring ribonucleotides or deoxyribonucleotides. Exemplary nucleotideanalogs are modified at any position so as to alter certain chemicalproperties of the nucleotide yet retain the ability of the nucleotideanalog to perform its intended function. Examples of positions of thenucleotide which may be derivatized include the 5 position, e.g.,5-(2-amino)propyl uridine, 5-bromo uridine, 5-propyne uridine,5-propenyl uridine, etc.; the 6 position, e.g., 6-(2-amino)propyluridine; the 8-position for adenosine and/or guanosines, e.g., 8-bromoguanosine, 8-chloro guanosine, 8-fluoroguanosine, etc. Nucleotideanalogs also include deaza nucleotides, e.g., 7-deaza-adenosine; O- andN-modified (e.g., alkylated, e.g., N6-methyl adenosine, or as otherwiseknown in the art) nucleotides; and other heterocyclically modifiednucleotide analogs such as those described in Herdewijn, AntisenseNucleic Acid Drug Dev., 2000 Aug. 10(4):297-310.

Nucleotide analogs may also comprise modifications to the sugar portionof the nucleotides. For example, the 2′ OH-group may be replaced by agroup selected from H, OR, R, F, Cl, Br, I, SH, SR, NH₂, NHR, NR₂, orCOOR, wherein R is substituted or unsubstituted C₁-C₆ alkyl, alkenyl,alkynyl, aryl, etc. Other possible modifications include those describedin U.S. Pat. Nos. 5,858,988, and 6,291,438.

The phosphate group of the nucleotide may also be modified, e.g., bysubstituting one or more of the oxygens of the phosphate group withsulfur (e.g., phosphorothioates), or by making other substitutions whichallow the nucleotide to perform its intended function such as describedin, for example, Eckstein, Antisense Nucleic Acid Drug Dev. 2000 Apr.10(2):117-21, Rusckowski et al. Antisense Nucleic Acid Drug Dev. 2000Oct. 10(5):333-45, Stein, Antisense Nucleic Acid Drug Dev. 2001 Oct.11(5): 317-25, Vorobjev et al. Antisense Nucleic Acid Drug Dev. 2001Apr. 11(2):77-85, and U.S. Pat. No. 5,684,143. Certain of theabove-referenced modifications (e.g., phosphate group modifications)preferably decrease the rate of hydrolysis of, for example,polynucleotides comprising said analogs in vivo or in vitro.

The term “oligonucleotide” refers to a short polymer of nucleotidesand/or nucleotide analogs. The term “RNA analog” refers to apolynucleotide (e.g., a chemically synthesized polynucleotide) having atleast one altered or modified nucleotide as compared to a correspondingunaltered or unmodified RNA but retaining the same or similar nature orfunction as the corresponding unaltered or unmodified RNA. As discussedabove, the oligonucleotides may be linked with linkages which result ina lower rate of hydrolysis of the RNA analog as compared to an RNAmolecule with phosphodiester linkages. For example, the nucleotides ofthe analog may comprise methylenediol, ethylene diol, oxymethylthio,oxyethylthio, oxycarbonyloxy, phosphorodiamidate, phosphoroamidate,and/or phosphorothioate linkages. Preferred RNA analogues include sugar-and/or backbone-modified ribonucleotides and/or deoxyribonucleotides.Such alterations or modifications can further include addition ofnon-nucleotide material, such as to the end(s) of the RNA or internally(at one or more nucleotides of the RNA). An RNA analog need only besufficiently similar to natural RNA that it has the ability to mediate(mediates) RNA interference.

As used herein, the term “RNA interference” (“RNAi”) refers to aselective intracellular degradation of RNA. RNAi occurs in cellsnaturally to remove foreign RNAs (e.g., viral RNAs). Natural RNAiproceeds via fragments cleaved from free dsRNA which direct thedegradative mechanism to other similar RNA sequences. Alternatively,RNAi can be initiated by the hand of man, for example, to silence theexpression of target genes.

An RNAi agent, e.g., an RNA silencing agent, having a strand which is“sequence sufficiently complementary to a target mRNA sequence to directtarget-specific RNA interference (RNAi)” means that the strand has asequence sufficient to trigger the destruction of the target mRNA by theRNAi machinery or process.

As used herein, the term “isolated RNA” (e.g., “isolated siRNA” or“isolated siRNA precursor”) refers to RNA molecules which aresubstantially free of other cellular material, or culture medium whenproduced by recombinant techniques, or substantially free of chemicalprecursors or other chemicals when chemically synthesized.

As used herein, the term “RNA silencing” refers to a group ofsequence-specific regulatory mechanisms (e.g. RNA interference (RNAi),transcriptional gene silencing (TGS), post-transcriptional genesilencing (PTGS), quelling, co-suppression, and translationalrepression) mediated by RNA molecules which result in the inhibition or“silencing” of the expression of a corresponding protein-coding gene.RNA silencing has been observed in many types of organisms, includingplants, animals, and fungi.

The term “discriminatory RNA silencing” refers to the ability of an RNAmolecule to substantially inhibit the expression of a “first” or“target” polynucleotide sequence while not substantially inhibiting theexpression of a “second” or “non-target” polynucleotide sequence,” e.g.,when both polynucleotide sequences are present in the same cell. In someembodiments, the target polynucleotide sequence corresponds to a targetgene, while the non-target polynucleotide sequence corresponds to anon-target gene. In other embodiments, the target polynucleotidesequence corresponds to a target allele, while the non-targetpolynucleotide sequence corresponds to a non-target allele. In someembodiments, the target polynucleotide sequence is the DNA sequenceencoding the regulatory region (e.g. promoter or enhancer elements) of atarget gene. In other embodiments, the target polynucleotide sequence isa target mRNA encoded by a target gene.

The term “in vitro” has its art recognized meaning, e.g., involvingpurified reagents or extracts, e.g., cell extracts. The term “in vivo”also has its art recognized meaning, e.g., involving living cells, e.g.,immortalized cells, primary cells, cell lines, and/or cells in anorganism.

As used herein, the term “transgene” refers to any nucleic acidmolecule, which is inserted by artifice into a cell, and becomes part ofthe genome of the organism that develops from the cell. Such a transgenemay include a gene that is partly or entirely heterologous (i.e.,foreign) to the transgenic organism, or may represent a gene homologousto an endogenous gene of the organism. The term “transgene” also means anucleic acid molecule that includes one or more selected nucleic acidsequences, e.g., DNAs, that encode one or more engineered RNAprecursors, to be expressed in a transgenic organism, e.g., animal,which is partly or entirely heterologous, i.e., foreign, to thetransgenic animal, or homologous to an endogenous gene of the transgenicanimal, but which is designed to be inserted into the animal's genome ata location which differs from that of the natural gene. A transgeneincludes one or more promoters and any other DNA, such as introns,necessary for expression of the selected nucleic acid sequence, alloperably linked to the selected sequence, and may include an enhancersequence.

A gene “involved” in a disease or disorder includes a gene, the normalor aberrant expression or function of which effects or causes thedisease or disorder or at least one symptom of said disease or disorder.

The term “gain-of-function mutation” as used herein, refers to anymutation in a gene in which the protein encoded by said gene (i.e., themutant protein) acquires a function not normally associated with theprotein (i.e., the wild type protein) causes or contributes to a diseaseor disorder. The gain-of-function mutation can be a deletion, addition,or substitution of a nucleotide or nucleotides in the gene which givesrise to the change in the function of the encoded protein. In oneembodiment, the gain-of-function mutation changes the function of themutant protein or causes interactions with other proteins. In anotherembodiment, the gain-of-function mutation causes a decrease in orremoval of normal wild-type protein, for example, by interaction of thealtered, mutant protein with said normal, wild-type protein.

As used herein, the term “target gene” is a gene whose expression is tobe substantially inhibited or “silenced.” This silencing can be achievedby RNA silencing, e.g., by cleaving the mRNA of the target gene ortranslational repression of the target gene. The term “non-target gene”is a gene whose expression is not to be substantially silenced. In oneembodiment, the polynucleotide sequences of the target and non-targetgene (e.g. mRNA encoded by the target and non-target genes) can differby one or more nucleotides. In another embodiment, the target andnon-target genes can differ by one or more polymorphisms (e.g., SingleNucleotide Polymorphisms or SNPs). In another embodiment, the target andnon-target genes can share less than 100% sequence identity. In anotherembodiment, the non-target gene may be a homologue (e.g. an orthologueor paralogue) of the target gene.

A “target allele” is an allele (e.g., a SNP allele) whose expression isto be selectively inhibited or “silenced.” This silencing can beachieved by RNA silencing, e.g., by cleaving the mRNA of the target geneor target allele by a siRNA. The term “non-target allele” is an allelewhose expression is not to be substantially silenced. In someembodiments, the target and non-target alleles can correspond to thesame target gene. In other embodiments, the target allele correspondsto, or is associated with, a target gene, and the non-target allelecorresponds to, or is associated with, a non-target gene. In oneembodiment, the polynucleotide sequences of the target and non-targetalleles can differ by one or more nucleotides. In another embodiment,the target and non-target alleles can differ by one or more allelicpolymorphisms (e.g., one or more SNPs). In another embodiment, thetarget and non-target alleles can share less than 100% sequenceidentity.

The term “polymorphism” as used herein, refers to a variation (e.g., oneor more deletions, insertions, or substitutions) in a gene sequence thatis identified or detected when the same gene sequence from differentsources or subjects (but from the same organism) are compared. Forexample, a polymorphism can be identified when the same gene sequencefrom different subjects are compared. Identification of suchpolymorphisms is routine in the art, the methodologies being similar tothose used to detect, for example, breast cancer point mutations.Identification can be made, for example, from DNA extracted from asubject's lymphocytes, followed by amplification of polymorphic regionsusing specific primers to said polymorphic region. Alternatively, thepolymorphism can be identified when two alleles of the same gene arecompared. In particular embodiments, the polymorphism is a singlenucleotide polymorphism (SNP).

A variation in sequence between two alleles of the same gene within anorganism is referred to herein as an “allelic polymorphism.” In someembodiments, the allelic polymorphism corresponds to a SNP allele. Forexample, the allelic polymorphism may comprise a single nucleotidevariation between the two alleles of a SNP. The polymorphism can be at anucleotide within a coding region but, due to the degeneracy of thegenetic code, no change in amino acid sequence is encoded.Alternatively, polymorphic sequences can encode a different amino acidat a particular position, but the change in the amino acid does notaffect protein function. Polymorphic regions can also be found innon-encoding regions of the gene. In exemplary embodiments, thepolymorphism is found in a coding region of the gene or in anuntranslated region (e.g., a 5′ UTR or 3′ UTR) of the gene.

As used herein, the term “allelic frequency” is a measure (e.g.,proportion or percentage) of the relative frequency of an allele (e.g.,a SNP allele) at a single locus in a population of individuals. Forexample, where a population of individuals carry n loci of a particularchromosomal locus (and the gene occupying the locus) in each of theirsomatic cells, then the allelic frequency of an allele is the fractionor percentage of loci that the allele occupies within the population. Inparticular embodiments, the allelic frequency of an allele (e.g., an SNPallele) is at least 10% (e.g., at least 15%, 20%, 25%, 30%, 35%, 40% ormore) in a sample population.

As used herein, the term “sample population” refers to a population ofindividuals comprising a statistically significant number ofindividuals. For example, the sample population may comprise 50, 75,100, 200, 500, 1000 or more individuals. In particular embodiments, thesample population may comprise individuals which share at least oncommon disease phenotype (e.g., a gain-of-function disorder) or mutation(e.g., a gain-of-function mutation).

As used herein, the term “heterozygosity” refers to the fraction ofindividuals within a population that are heterozygous (e.g., contain twoor more different alleles) at a particular locus (e.g., at a SNP).Heterozygosity may be calculated for a sample population using methodsthat are well known to those skilled in the art.

The term “polyglutamine domain,” as used herein, refers to a segment ordomain of a protein that consist of consecutive glutamine residueslinked to peptide bonds. In one embodiment the consecutive regionincludes at least 5 glutamine residues.

The term “expanded polyglutamine domain” or “expanded polyglutaminesegment,” as used herein, refers to a segment or domain of a proteinthat includes at least 35 consecutive glutamine residues linked bypeptide bonds. Such expanded segments are found in subjects afflictedwith a polyglutamine disorder, as described herein, whether or not thesubject has shown to manifest symptoms.

The term “trinucleotide repeat” or “trinucleotide repeat region” as usedherein, refers to a segment of a nucleic acid sequence e.g.,) thatconsists of consecutive repeats of a particular trinucleotide sequence.In one embodiment, the trinucleotide repeat includes at leastconsecutive trinucleotide sequences. Exemplary trinucleotide sequencesinclude, but are not limited to, CAG, CGG, GCC, GAA, CTG and/or CGG.

The term “trinucleotide repeat diseases” as used herein, refers to anydisease or disorder characterized by an expanded trinucleotide repeatregion located within a gene, the expanded trinucleotide repeat regionbeing causative of the disease or disorder. Examples of trinucleotiderepeat diseases include, but are not limited to spino-cerebellar ataxiatype 12 spino-cerebellar ataxia type 8, fragile X syndrome, fragile XEmental retardation, Friedreich's ataxia and myotonic dystrophy.Exemplary trinucleotide repeat diseases for treatment according to thepresent invention are those characterized or caused by an expandedtrinucleotide repeat region at the 5′ end of the coding region of agene, the gene encoding a mutant protein which causes or is causative ofthe disease or disorder. Certain trinucleotide diseases, for example,fragile X syndrome, where the mutation is not associated with a codingregion may not be suitable for treatment according to the methodologiesof the present invention, as there is no suitable mRNA to be targeted byRNAi. By contrast, disease such as Friedreich's ataxia may be suitablefor treatment according to the methodologies of the invention because,although the causative mutation is not within a coding region (i.e.,lies within an intron), the mutation may be within, for example, an mRNAprecursor (e.g., a pre-spliced mRNA precursor).

The phrase “examining the function of a gene in a cell or organism”refers to examining or studying the expression, activity, function orphenotype arising therefrom.

As used herein, the term “RNA silencing agent” refers to an RNA which iscapable of inhibiting or “silencing” the expression of a target gene. Insome embodiments, the RNA silencing agent is capable of preventingcomplete processing (e.g., the full translation and/or expression) of amRNA molecule through a post-transcriptional silencing mechanism. RNAsilencing agents include small (<50 b.p.), noncoding RNA molecules, forexample RNA duplexes comprising paired strands, as well as precursorRNAs from which such small non-coding RNAs can be generated. ExemplaryRNA silencing agents include siRNAs, miRNAs, siRNA-like duplexes,antisense oligonucleotides, GAPMER molecules, and dual-functionoligonucleotides as well as precursors thereof. In one embodiment, theRNA silencing agent is capable of inducing RNA interference. In anotherembodiment, the RNA silencing agent is capable of mediatingtranslational repression.

As used herein, the term “rare nucleotide” refers to a naturallyoccurring nucleotide that occurs infrequently, including naturallyoccurring deoxyribonucleotides or ribonucleotides that occurinfrequently, e.g., a naturally occurring ribonucleotide that is notguanosine, adenosine, cytosine, or uridine. Examples of rare nucleotidesinclude, but are not limited to, inosine, 1-methyl inosine,pseudouridine, 5,6-dihydrouridine, ribothymidine, 2N-methylguanosine and2,2N,N-dimethylguanosine.

The term “engineered,” as in an engineered RNA precursor, or anengineered nucleic acid molecule, indicates that the precursor ormolecule is not found in nature, in that all or a portion of the nucleicacid sequence of the precursor or molecule is created or selected by ahuman. Once created or selected, the sequence can be replicated,translated, transcribed, or otherwise processed by mechanisms within acell. Thus, an RNA precursor produced within a cell from a transgenethat includes an engineered nucleic acid molecule is an engineered RNAprecursor.

As used herein, the term “microRNA” (“miRNA”), also referred to in theart as “small temporal RNAs” (“stRNAs”), refers to a small (10-50nucleotide) RNA which are genetically encoded (e.g., by viral,mammalian, or plant genomes) and are capable of directing or mediatingRNA silencing. An “miRNA disorder” shall refer to a disease or disordercharacterized by an aberrant expression or activity of an miRNA.

As used herein, the term “dual functional oligonucleotide” refers to aRNA silencing agent having the formula T-L-μ, wherein T is an mRNAtargeting moiety, L is a linking moiety, and is a miRNA recruitingmoiety. As used herein, the terms “mRNA targeting moiety,” “targetingmoiety,” “mRNA targeting portion” or “targeting portion” refer to adomain, portion or region of the dual functional oligonucleotide havingsufficient size and sufficient complementarity to a portion or region ofan mRNA chosen or targeted for silencing (i.e., the moiety has asequence sufficient to capture the target mRNA). As used herein, theterm “linking moiety” or “linking portion” refers to a domain, portionor region of the RNA-silencing agent which covalently joins or links themRNA.

As used herein, the term “antisense strand” of an RNA silencing agent,e.g., an siRNA or RNA silencing agent, refers to a strand that issubstantially complementary to a section of about 10-50 nucleotides,e.g., about 15-30, 16-25, 18-23 or 19-22 nucleotides of the mRNA of thegene targeted for silencing. The antisense strand or first strand hassequence sufficiently complementary to the desired target mRNA sequenceto direct target-specific silencing, e.g., complementarity sufficient totrigger the destruction of the desired target mRNA by the RNAi machineryor process (RNAi interference) or complementarity sufficient to triggertranslational repression of the desired target mRNA.

The term “sense strand” or “second strand” of an RNA silencing agent,e.g., an siRNA or RNA silencing agent, refers to a strand that iscomplementary to the antisense strand or first strand. Antisense andsense strands can also be referred to as first or second strands, thefirst or second strand having complementarity to the target sequence andthe respective second or first strand having complementarity to saidfirst or second strand. miRNA duplex intermediates or siRNA-likeduplexes include a miRNA strand having sufficient complementarity to asection of about 10-50 nucleotides of the mRNA of the gene targeted forsilencing and a miRNA* strand having sufficient complementarity to forma duplex with the miRNA strand.

As used herein, the term “guide strand” refers to a strand of an RNAsilencing agent, e.g., an antisense strand of an siRNA duplex or siRNAsequence, that enters into the RISC complex and directs cleavage of thetarget mRNA.

As used herein, the term “asymmetry,” as in the asymmetry of the duplexregion of an RNA silencing agent (e.g., the stem of an shRNA), refers toan inequality of bond strength or base pairing strength between thetermini of the RNA silencing agent (e.g., between terminal nucleotideson a first strand or stem portion and terminal nucleotides on anopposing second strand or stem portion), such that the 5′ end of onestrand of the duplex is more frequently in a transient unpaired, e.g.,single-stranded, state than the 5′ end of the complementary strand. Thisstructural difference determines that one strand of the duplex ispreferentially incorporated into a RISC complex. The strand whose 5′ endis less tightly paired to the complementary strand will preferentiallybe incorporated into RISC and mediate RNAi.

As used herein, the term “bond strength” or “base pair strength” refersto the strength of the interaction between pairs of nucleotides (ornucleotide analogs) on opposing strands of an oligonucleotide duplex(e.g., an siRNA duplex), due primarily to H-bonding, van der Waalsinteractions, and the like between said nucleotides (or nucleotideanalogs).

As used herein, the “5′ end,” as in the 5′ end of an antisense strand,refers to the 5′ terminal nucleotides, e.g., between one and about 5nucleotides at the 5′ terminus of the antisense strand. As used herein,the “3′ end,” as in the 3′ end of a sense strand, refers to the region,e.g., a region of between one and about 5 nucleotides, that iscomplementary to the nucleotides of the 5′ end of the complementaryantisense strand.

As used herein the term “destabilizing nucleotide” refers to a firstnucleotide or nucleotide analog capable of forming a base pair withsecond nucleotide or nucleotide analog such that the base pair is oflower bond strength than a conventional base pair (i.e., Watson-Crickbase pair). In some embodiments, the destabilizing nucleotide is capableof forming a mismatch base pair with the second nucleotide. In otherembodiments, the destabilizing nucleotide is capable of forming a wobblebase pair with the second nucleotide. In yet other embodiments, thedestabilizing nucleotide is capable of forming an ambiguous base pairwith the second nucleotide.

As used herein, the term “base pair” refers to the interaction betweenpairs of nucleotides (or nucleotide analogs) on opposing strands of anoligonucleotide duplex (e.g., a duplex formed by a strand of a RNAsilencing agent and a target mRNA sequence), due primarily to H-bonding,van der Waals interactions, and the like between said nucleotides (ornucleotide analogs). As used herein, the term “bond strength” or “basepair strength” refers to the strength of the base pair.

As used herein, the term “mismatched base pair” refers to a base pairconsisting of non-complementary or non-Watson-Crick base pairs, forexample, not normal complementary G:C, A:T or A:U base pairs. As usedherein the term “ambiguous base pair” (also known as anon-discriminatory base pair) refers to a base pair formed by auniversal nucleotide.

As used herein, term “universal nucleotide” (also known as a “neutralnucleotide”) include those nucleotides (e.g. certain destabilizingnucleotides) having a base (a “universal base” or “neutral base”) thatdoes not significantly discriminate between bases on a complementarypolynucleotide when forming a base pair. Universal nucleotides arepredominantly hydrophobic molecules that can pack efficiently intoantiparallel duplex nucleic acids (e.g., double-stranded DNA or RNA) dueto stacking interactions. The base portion of universal nucleotidestypically comprise a nitrogen-containing aromatic heterocyclic moiety.

As used herein, the terms “sufficient complementarity” or “sufficientdegree of complementarity” mean that the RNA silencing agent has asequence (e.g. in the antisense strand, mRNA targeting moiety or miRNArecruiting moiety) which is sufficient to bind the desired target RNA,respectively, and to trigger the RNA silencing of the target mRNA.

As used herein, the term “translational repression” refers to aselective inhibition of mRNA translation. Natural translationalrepression proceeds via miRNAs cleaved from shRNA precursors. Both RNAiand translational repression are mediated by RISC. Both RNAi andtranslational repression occur naturally or can be initiated by the handof man, for example, to silence the expression of target genes.

As used herein, the term “alkoxy,” refers to the group —O-alkyl, whereinalkyl is as defined herein. Alkoxy includes, by way of example, methoxy,ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, t-butoxy and thelike. In some embodiments, C₁-C₆ alkoxy groups are provided herein.

As used herein, the term “halo” or “halogen” alone or as part of anothersubstituent means, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom, preferably, fluorine, chlorine, or bromine,more preferably, fluorine or chlorine.

As used herein, the term “hydroxy” alone or as part of anothersubstituent means, unless otherwise stated, an alcohol moiety having theformula —OH.

Preparation of linkers can involve the protection and deprotection ofvarious chemical groups. The need for protection and deprotection, andthe selection of appropriate protecting groups can be readily determinedby one skilled in the art. The chemistry of protecting groups can befound, for example, in Greene, et al., Protective Groups in OrganicSynthesis, 4d. Ed., Wiley & Sons, 2007, which is incorporated herein byreference in its entirety. Adjustments to the protecting groups andformation and cleavage methods described herein may be adjusted asnecessary in light of the various substituents.

Various methodologies of the instant invention include step thatinvolves comparing a value, level, feature, characteristic, property,etc. to a “suitable control,” referred to interchangeably herein as an“appropriate control.” A “suitable control” or “appropriate control” isany control or standard familiar to one of ordinary skill in the artuseful for comparison purposes. In one embodiment, a “suitable control”or “appropriate control” is a value, level, feature, characteristic,property, etc. determined prior to performing an RNAi methodology, asdescribed herein. For example, a transcription rate, mRNA level,translation rate, protein level, biological activity, cellularcharacteristic or property, genotype, phenotype, etc. can be determinedprior to introducing an RNA silencing agent of the invention into a cellor organism. In another embodiment, a “suitable control” or “appropriatecontrol” is a value, level, feature, characteristic, property, etc.determined in a cell or organism, e.g., a control or normal cell ororganism, exhibiting, for example, normal traits. In yet anotherembodiment, a “suitable control” or “appropriate control” is apredefined value, level, feature, characteristic, property, etc.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and example are illustrative only and not intendedto be limiting.

Various aspects of the invention are described in further detail in thefollowing subsections.

I. Novel Modified Oligonucleotides

In one aspect, the disclosure provides a modified oligonucleotide, saidoligonucleotide having a 5′ end, a 3′ end, that is complementary to atarget, wherein the oligonucleotide comprises a sense and antisensestrand, and at least one modified intersubunit linkage of Formula (I):

wherein:

each B is, independently, a base pairing moiety;

W is selected from the group consisting of O, OCH₂, OCH, CH₂, and CH;

each X is, independently, selected from the group consisting of halo(e.g., fluoro or chloro), hydroxy, and C₁₋₆ alkoxy;

Y is selected from the group consisting of O⁻, OH, OR, NH⁻, NH₂, S⁻, andSH;

Z is selected from the group consisting of O and CH₂;

R is a protecting group; and

is an optional double bond.

In some embodiments of Formula (I), when Y is O⁻, either Z or W is notO.

In some embodiments of Formula (I), Z is CH₂ and W is CH₂. In anotherembodiment, the modified intersubunit linkage of Formula (I) is amodified intersubunit linkage of Formula (II):

In some embodiments of Formula (I), Z is CH₂ and W is O. In anotherembodiment, wherein the modified intersubunit linkage of Formula (I) isa modified intersubunit linkage of Formula (III):

In some embodiments of Formula (I), Z is O and W is CH₂. In anotherembodiment, the modified intersubunit linkage of Formula (I) is amodified intersubunit linkage of Formula (IV):

In some embodiments of Formula (I), Z is O and W is CH. In anotherembodiment, the modified intersubunit linkage of Formula (I) is amodified intersubunit linkage of Formula V:

In some embodiments, the modified intersubunit linkage of Formula I is amodified intersubunit linkage of Formula VI:

In some embodiments of Formula VI:

-   -   each B is, independently, a base pairing moiety;    -   each X is, independently, selected from the group consisting of        halo, hydroxy, and C₁₋₆ alkoxy; optionally wherein each X is,        independently, selected from the group consisting of halo (e.g.,        fluoro) and C₁₋₆ alkoxy (e.g., methoxy, ethoxy, n-propoxy,        sec-propoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, or        n-heptoxy);    -   Y is selected from the group consisting of O⁻, OH, OR, NH⁻, NH₂,        S⁻, and SH, optionally wherein Y is selected from the group        consisting of O⁻, OH, and OR;    -   Z is selected from the group consisting of O and CH₂; and    -   is an optional double bond.

In some embodiments of Formula VI:

-   -   each X is, independently, selected from the group consisting of        fluoro, hydroxy, and C₁₋₆ alkoxy; optionally wherein each X is,        independently, selected from the group consisting of fluoro and        C₁₋₆ alkoxy (e.g., methoxy, ethoxy, n-propoxy, sec-propoxy,        n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, or n-heptoxy);    -   Y is selected from the group consisting of O⁻, OH, and OR;    -   Z is selected from the group consisting of O and CH₂; and    -   is an optional double bond.

In some embodiments of Formula VI:

-   -   each X is, independently, selected from the group consisting of        fluoro, hydroxy, methoxy, ethoxy, n-propoxy, sec-propoxy,        n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, and n-heptoxy;    -   Y is selected from the group consisting of O⁻, OH, and OR;    -   Z is selected from the group consisting of O and CH₂; and    -   is an optional double bond.

In some embodiments of Formula VI:

-   -   each X is, independently, selected from the group consisting of        fluoro, hydroxy, methoxy, ethoxy, n-propoxy, sec-propoxy,        n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, and n-heptoxy;    -   Y is selected from the group consisting of O⁻, OH, and OR;    -   Z is O; and    -   is an optional double bond.

In some embodiments of Formula VI:

-   -   each X is, independently, selected from the group consisting of        fluoro, hydroxy, methoxy, ethoxy, n-propoxy, sec-propoxy,        n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, and n-heptoxy;    -   Y is selected from the group consisting of O⁻, OH, and OR;    -   Z is CH₂; and    -   is an optional double bond.

In some embodiments of Formula VI:

-   -   each X is, independently, selected from the group consisting of        fluoro, hydroxy, and methoxy;    -   Y is selected from the group consisting of O⁻, OH, and OR;    -   Z is O; and    -   is an optional double bond.

In some embodiments of Formula VI:

-   -   each X is, independently, selected from the group consisting of        fluoro, hydroxy, and methoxy;    -   Y is selected from the group consisting of O⁻, OH, and OR;    -   Z is CH₂; and    -   is an optional double bond.

In some embodiments of Formula (I), Z is O and W is OCH₂. In anotherembodiment, the modified intersubunit linkage of Formula (I) is amodified intersubunit linkage of Formula VIa:

In some embodiments of Formula (I), Z is CH₂ and W is CH. In anotherembodiment, the modified intersubunit linkage of Formula (I) is amodified intersubunit linkage of Formula VII:

In some embodiments of Formula (I), the base pairing moiety B isselected from the group consisting of adenine, guanine, cytosine, anduracil.

In some embodiments, the modified oligonucleotide is incorporated intosiRNA, said modified siRNA having a 5′ end, a 3′ end, that iscomplementary to a target, wherein the siRNA comprises a sense andantisense strand, and at least one modified intersubunit linkage ofFormula (I):

wherein:

each B is, independently, a base pairing moiety;

W is selected from the group consisting of O, OCH₂, OCH, CH₂, and CH;

each X is, independently, selected from the group consisting of halo(e.g., fluoro or chloro), hydroxy, and C₁₋₆ alkoxy;

Y is selected from the group consisting of O⁻, OH, OR, NH⁻, NH₂, S⁻, andSH;

Z is selected from the group consisting of O and CH₂;

R is a protecting group; and

is an optional double bond.

In some embodiments of Formula (I), when Y is O⁻, either Z or W is notO. In another embodiment of Formula (I), Z is CH₂ and W is CH₂.

In yet another embodiment, the modified intersubunit linkage of Formula(I) is a modified intersubunit linkage of Formula (II):

In some embodiments of Formula (I), Z is CH₂ and W is O. In anotherembodiment, the modified intersubunit linkage of Formula (I) is amodified intersubunit linkage of Formula (III):

In some embodiments of Formula (I), Z is O and W is CH₂. In anotherembodiment, the modified intersubunit linkage of Formula (I) is amodified intersubunit linkage of Formula (IV):

In some embodiments of Formula (I), Z is O and W is CH. In anotherembodiment, the modified intersubunit linkage of Formula (I) is amodified intersubunit linkage of Formula V:

In some embodiments of Formula (I), wherein Z is CH═CH and W is CH₂. Inanother embodiment, the modified intersubunit linkage of Formula (I) isa modified intersubunit linkage of Formula VII:

In some embodiments, the modified intersubunit linkage of Formula I is amodified intersubunit linkage of Formula VI:

In some embodiments of Formula VI:

-   -   each B is, independently, a base pairing moiety;    -   each X is, independently, selected from the group consisting of        halo, hydroxy, and C₁₋₆ alkoxy; optionally wherein each X is,        independently, selected from the group consisting of halo (e.g.,        fluoro) and C₁₋₆ alkoxy (e.g., methoxy, ethoxy, n-propoxy,        sec-propoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, or        n-heptoxy);    -   Y is selected from the group consisting of O⁻, OH, OR, NH⁻, NH₂,        S⁻, and SH, optionally wherein Y is selected from the group        consisting of O⁻, OH, and OR;    -   Z is selected from the group consisting of O and CH₂; and    -   is an optional double bond.

In some embodiments of Formula VI:

-   -   each X is, independently, selected from the group consisting of        fluoro, hydroxy, and C₁₋₆ alkoxy; optionally wherein each X is,        independently, selected from the group consisting of fluoro and        C₁₋₆ alkoxy (e.g., methoxy, ethoxy, n-propoxy, sec-propoxy,        n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, or n-heptoxy);    -   Y is selected from the group consisting of O⁻, OH, and OR;    -   Z is selected from the group consisting of O and CH₂; and    -   is an optional double bond.

In some embodiments of Formula VI:

-   -   each X is, independently, selected from the group consisting of        fluoro, hydroxy, methoxy, ethoxy, n-propoxy, sec-propoxy,        n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, and n-heptoxy;    -   Y is selected from the group consisting of O⁻, OH, and OR;    -   Z is selected from the group consisting of O and CH₂; and    -   is an optional double bond.

In some embodiments of Formula VI:

-   -   each X is, independently, selected from the group consisting of        fluoro, hydroxy, methoxy, ethoxy, n-propoxy, sec-propoxy,        n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, and n-heptoxy;    -   Y is selected from the group consisting of O⁻, OH, and OR;    -   Z is O; and    -   is an optional double bond.

In some embodiments of Formula VI:

-   -   each X is, independently, selected from the group consisting of        fluoro, hydroxy, methoxy, ethoxy, n-propoxy, sec-propoxy,        n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, and n-heptoxy;    -   Y is selected from the group consisting of O⁻, OH, and OR;    -   Z is CH₂; and    -   is an optional double bond.

In some embodiments of Formula VI:

-   -   each X is, independently, selected from the group consisting of        fluoro, hydroxy, and methoxy;    -   Y is selected from the group consisting of O⁻, OH, and OR;    -   Z is O; and    -   is an optional double bond.

In some embodiments of Formula VI:

-   -   each X is, independently, selected from the group consisting of        fluoro, hydroxy, and methoxy;    -   Y is selected from the group consisting of O⁻, OH, and OR;    -   Z is CH₂; and    -   is an optional double bond.

In some embodiments of Formula (I), Z is O and W is OCH₂. In anotherembodiment, the modified intersubunit linkage of Formula (I) is amodified intersubunit linkage of Formula VIa:

In some embodiments of Formula (I), wherein Z is CH₂ and W is CH. Inanother embodiment, the modified intersubunit linkage of Formula (I) isa modified intersubunit linkage of Formula VII:

In some embodiments, the base pairing moiety B is selected from thegroup consisting of adenine, guanine, cytosine, and uracil. In anotherembodiment, B is adenine. In yet another embodiment, B is guanine. Instill another embodiment, B is cytosine. In some embodiments, B isuracil.

In some embodiments, the modified oligonucleotide is incorporated intosiRNA, said modified siRNA having a 5′ end, a 3′ end, that iscomplementary to a target, wherein the siRNA comprises a sense andantisense strand, and at least one modified intersubunit linkage ofFormula (I):

wherein:

each B is, independently, a base pairing moiety;

W is selected from the group consisting of O, OCH₂, OCH, CH₂, and CH;

each X is, independently, selected from the group consisting of halo(e.g., fluoro or chloro), hydroxy, and C₁₋₆ alkoxy;

Y is selected from the group consisting of O⁻, OH, OR, NH⁻, NH₂, S⁻, andSH;

Z is selected from the group consisting of O and CH₂;

R is a protecting group selected from the group consisting ofdimethoxytrityl (DMTr), succinate, tert-butyl dimethylsilyl (TBDMS),benzoyl (Bz), benzyl (Bn), methoxyethoxymethyl ether (MOM),methoxybenzyl ether (PMB), methylthiomethyl ether, pivaloyl (Piv),tetrahydropyranyl (THP), tetrahydrofuranyl (THF), trityl (Trt),triisopropylsilyl (TIPS), tert-butyldiphenylsilyl (TBDPS), and acetate;and

is an optional double bond.

In some embodiments, the modified oligonucleotide is incorporated intosiRNA, said modified siRNA having a 5′ end, a 3′ end, that iscomplementary to a target and comprises a sense and antisense strand,wherein the siRNA comprises at least one modified intersubunit linkageis of Formula VIII:

wherein:

D is selected from the group consisting of O, OCH₂, OCH, CH₂, and CH;

C is selected from the group consisting of O⁻, OH, OR¹, NH⁻, NH₂, S⁻,and SH;

A is selected from the group consisting of O and CH₂;

R¹ is a protecting group;

is an optional double bond; and

the intersubunit is bridging two optionally modified nucleosides.

In some embodiments, when C is O⁻, either A or D is not O.

In some embodiments, D is CH₂. In another embodiment, the modifiedintersubunit linkage of Formula VIII is a modified intersubunit linkageof Formula (IX):

In some embodiments, D is O. In another embodiment, the modifiedintersubunit linkage of Formula VIII is a modified intersubunit linkageof Formula (X):

In some embodiments, D is CH₂. In another embodiment, the modifiedintersubunit linkage of Formula (VIII) is a modified intersubunitlinkage of Formula (XI):

In some embodiments, D is CH. In another embodiment, the modifiedintersubunit linkage of Formula VIII is a modified intersubunit linkageof Formula (XII):

In another embodiment, the modified intersubunit linkage of Formula(VII) is a modified intersubunit linkage of Formula (XIV):

In some embodiments, D is OCH₂. In another embodiment, the modifiedintersubunit linkage of Formula (VII) is a modified intersubunit linkageof Formula (XIII):

In another embodiment, the modified intersubunit linkage of Formula(VII) is a modified intersubunit linkage of Formula (XXa):

In some embodiments of the modified siRNA linkage, each optionallymodified nucleoside is independently, at each occurrence, selected fromthe group consisting of adenosine, guanosine, cytidine, and uridine.

In some embodiments, the modified oligonucleotide is incorporated intosiRNA, said modified siRNA having a 5′ end, a 3′ end, that iscomplementary to a target and comprises a sense and antisense strand,wherein the siRNA comprises at least one modified intersubunit linkageis of Formula (VIII):

wherein:

D is selected from the group consisting of O, OCH₂, OCH, CH₂, and CH;

C is selected from the group consisting of O, OH, OR¹, NH⁻, NH₂, S⁻, andSH;

A is selected from the group consisting of O and CH₂;

R¹ is a protecting group selected from the group consisting ofdimethoxytrityl (DMTr), succinate, tert-butyl dimethylsilyl (TBDMS),benzoyl (Bz), benzyl (Bn), methoxyethoxymethyl ether (MOM),methoxybenzyl ether (PMB), methylthiomethyl ether, pivaloyl (Piv),tetrahydropyranyl (THP), tetrahydrofuranyl (THF), trityl (Trt),triisopropylsilyl (TIPS), tert-butyldiphenylsilyl (TBDPS), and acetate;

is an optional double bond; and

-   -   the intersubunit is bridging two optionally modified        nucleosides.

In certain exemplary embodiments of Formula (I), W is O. In anotherembodiment, W is CH₂. In yet another embodiment, W is CH.

In certain exemplary embodiments of Formula (I), X is OH. In anotherembodiment, X is OCH₃. In yet another embodiment, X is halo.

In a certain embodiment of Formula (I), the modified siRNA does notcomprise a 2′-fluoro substituent.

In some embodiments of Formula (I), Y is O⁻. In another embodiment, Y isOH. In yet another embodiment, Y is OR. In still another embodiment, Yis NH⁻. In some embodiments, Y is NH₂. In another embodiment, Y is S⁻.In yet another embodiment, Y is SH.

In some embodiments of Formula (I), Z is O. In another embodiment, Z isCH₂.

In some embodiments, the modified intersubunit linkage is inserted onposition 1-2 of the antisense strand. In another embodiment, themodified intersubunit linkage is inserted on position 6-7 of theantisense strand. In yet another embodiment, the modified intersubunitlinkage is inserted on position 10-11 of the antisense strand. In stillanother embodiment, the modified intersubunit linkage is inserted onposition 19-20 of the antisense strand. In some embodiments, themodified intersubunit linkage is inserted on positions 5-6 and 18-19 ofthe antisense strand.

In some embodiments, R is DMTr. In another embodiment, R is succinate.In yet another embodiment, R is TBDPS. In still another embodiment, R isacetate.

In an exemplary embodiment of the modified siRNA linkage of Formula(VIII), C is O⁻. In another embodiment, C is OH. In yet anotherembodiment, C is OR¹. In still another embodiment, C is NH⁻. In someembodiments, C is NH₂. In another embodiment, C is S⁻. In yet anotherembodiment, C is SH.

In an exemplary embodiment of the modified siRNA linkage of Formula(VIII), A is O. In another embodiment, A is CH₂. In yet anotherembodiment, C is OR¹. In still another embodiment, C is NH⁻. In someembodiments, C is NH₂. In another embodiment, C is S⁻. In yet anotherembodiment, C is SH.

In a certain embodiment of the modified siRNA linkage of Formula (VIII),the optionally modified nucleoside is adenosine. In another embodimentof the modified siRNA linkage of Formula (VIII), the optionally modifiednucleoside is guanosine. In another embodiment of the modified siRNAlinkage of Formula (VIII), the optionally modified nucleoside iscytidine. In another embodiment of the modified siRNA linkage of Formula(VIII), the optionally modified nucleoside is uridine.

In some embodiments of the modified siRNA linkage, wherein the linkageis inserted on position 1-2 of the antisense strand. In anotherembodiment, the linkage is inserted on position 6-7 of the antisensestrand. In yet another embodiment, the linkage is inserted on position10-11 of the antisense strand. In still another embodiment, the linkageis inserted on position 19-20 of the antisense strand. In someembodiments, the linkage is inserted on positions 5-6 and 18-19 of theantisense strand.

In some embodiments of Formula (I), the base pairing moiety B isadenine. In some embodiments of Formula (I), the base pairing moiety Bis guanine. In some embodiments of Formula (I), the base pairing moietyB is cytosine. In some embodiments of Formula (I), the base pairingmoiety B is uracil.

In some embodiments of Formula (I), W is O. In some embodiments ofFormula (I), W is CH₂. In some embodiments of Formula (I), W is CH.

In some embodiments of Formula (I), X is OH. In some embodiments ofFormula (I), X is OCH₃. In some embodiments of Formula (I), X is halo.

In an exemplary embodiment of Formula (I), the modified oligonucleotidedoes not comprise a 2′-fluoro substituent.

In some embodiments of Formula (I), Y is O⁻. In some embodiments ofFormula (I), Y is OH. In some embodiments of Formula (I), Y is OR. Insome embodiments of Formula (I), Y is NH⁻. In some embodiments ofFormula (I), Y is NH₂. In some embodiments of Formula (I), Y is S⁻. Insome embodiments of Formula (I), Y is SH.

In some embodiments of Formula (I), Z is O. In some embodiments ofFormula (I), Z is CH₂.

In some embodiments of the Formula (I), the linkage is inserted onposition 1-2 of the antisense strand. In another embodiment of Formula(I), the linkage is inserted on position 6-7 of the antisense strand. Inyet another embodiment of Formula (I), the linkage is inserted onposition 10-11 of the antisense strand. In still another embodiment ofFormula (I), the linkage is inserted on position 19-20 of the antisensestrand. In some embodiments of Formula (I), the linkage is inserted onpositions 5-6 and 18-19 of the antisense strand.

In an aspect provided herein, is a method for preparing theoligonucleotides of the invention as summarized in FIG. 8.

In an aspect of the invention, the oligonucleotides and siRNA providedherein can be incorporated into a CRISPR/Cas system.

Genomic sequence for each target sequence can be found in, for example,the publicly available database maintained by the NCBI.

II. siRNA Design

In some embodiments, siRNAs are designed as follows. First, a portion ofthe target gene (e.g., a target gene of interest, such as the ApoEgene). Cleavage of mRNA at these sites should eliminate translation ofcorresponding protein. Sense strands were designed based on the targetsequence. Preferably the portion (and corresponding sense strand)includes about 19 to 25 nucleotides, e.g., 19, 20, 21, 22, 23, 24 or 25nucleotides. More preferably, the portion (and corresponding sensestrand) includes 21, 22 or 23 nucleotides. The skilled artisan willappreciate, however, that siRNAs having a length of less than 19nucleotides or greater than 25 nucleotides can also function to mediateRNAi. Accordingly, siRNAs of such length are also within the scope ofthe instant invention provided that they retain the ability to mediateRNAi. Longer RNAi agents have been demonstrated to elicit an interferonor PKR response in certain mammalian cells which may be undesirable.Preferably, the RNAi agents of the invention do not elicit a PKRresponse (i.e., are of a sufficiently short length). However, longerRNAi agents may be useful, for example, in cell types incapable ofgenerating a PKR response or in situations where the PKR response hasbeen down-regulated or dampened by alternative means.

The sense strand sequence is designed such that the target sequence isessentially in the middle of the strand. Moving the target sequence toan off-center position may, in some instances, reduce efficiency ofcleavage by the siRNA. Such compositions, i.e., less efficientcompositions, may be desirable for use if off-silencing of the wild-typemRNA is detected.

The antisense strand is routinely the same length as the sense strandand includes complementary nucleotides. In one embodiment, the strandsare fully complementary, i.e., the strands are blunt-ended when alignedor annealed. In another embodiment, the strands comprise align or annealsuch that 1-, 2-, 3-, 4-, 5-, 6- or 7-nucleotide overhangs aregenerated, i.e., the 3′ end of the sense strand extends 1, 2, 3, 4, 5, 6or 7 nucleotides further than the 5′ end of the antisense strand and/orthe 3′ end of the antisense strand extends 1, 2, 3, 4, 5, 6 or 7nucleotides further than the 5′ end of the sense strand. Overhangs cancomprise (or consist of) nucleotides corresponding to the target genesequence (or complement thereof). Alternatively, overhangs can comprise(or consist of) deoxyribonucleotides, for example dTs, or nucleotideanalogs, or other suitable non-nucleotide material.

To facilitate entry of the antisense strand into RISC (and thus increaseor improve the efficiency of target cleavage and silencing), the basepair strength between the 5′ end of the sense strand and 3′ end of theantisense strand can be altered, e.g., lessened or reduced, as describedin detail in U.S. Pat. Nos. 7,459,547, 7,772,203 and 7,732,593, entitled“Methods and Compositions for Controlling Efficacy of RNA Silencing”(filed Jun. 2, 2003) and U.S. Pat. Nos. 8,309,704, 7,750,144, 8,304,530,8,329,892 and 8,309,705, entitled “Methods and Compositions forEnhancing the Efficacy and Specificity of RNAi” (filed Jun. 2, 2003),the contents of which are incorporated in their entirety by thisreference. In one embodiment of these aspects of the invention, thebase-pair strength is less due to fewer G:C base pairs between the 5′end of the first or antisense strand and the 3′ end of the second orsense strand than between the 3′ end of the first or antisense strandand the 5′ end of the second or sense strand. In another embodiment, thebase pair strength is less due to at least one mismatched base pairbetween the 5′ end of the first or antisense strand and the 3′ end ofthe second or sense strand. In certain exemplary embodiments, themismatched base pair is selected from the group consisting of G:A, C:A,C:U, G:G, A:A, C:C and U:U. In another embodiment, the base pairstrength is less due to at least one wobble base pair, e.g., G:U,between the 5′ end of the first or antisense strand and the 3′ end ofthe second or sense strand. In another embodiment, the base pairstrength is less due to at least one base pair comprising a rarenucleotide, e.g., inosine (I). In certain exemplary embodiments, thebase pair is selected from the group consisting of an I:A, I:U and I:C.In yet another embodiment, the base pair strength is less due to atleast one base pair comprising a modified nucleotide. In certainexemplary embodiments, the modified nucleotide is selected from thegroup consisting of 2-amino-G, 2-amino-A, 2,6-diamino-G, and2,6-diamino-A.

The design of siRNAs suitable for targeting the target sequences ofinterest is described in detail below. siRNAs can be designed accordingto the above exemplary teachings for any other target sequences found inthe target gene. Moreover, the technology is applicable to targeting anyother target sequences, e.g., non-disease causing target sequences.

To validate the effectiveness by which siRNAs destroy mRNAs (e.g., mRNAexpressed from a target gene of interest), the siRNA can be incubatedwith cDNA (e.g., cDNA corresponding to a target gene of interest) in aDrosophila-based in vitro mRNA expression system. Radiolabeled with ³²P,newly synthesized mRNAs (e.g., target mRNA) are detectedautoradiographically on an agarose gel. The presence of cleaved mRNAindicates mRNA nuclease activity. Suitable controls include omission ofsiRNA. Alternatively, control siRNAs are selected having the samenucleotide composition as the selected siRNA, but without significantsequence complementarity to the appropriate target gene. Such negativecontrols can be designed by randomly scrambling the nucleotide sequenceof the selected siRNA; a homology search can be performed to ensure thatthe negative control lacks homology to any other gene in the appropriategenome. In addition, negative control siRNAs can be designed byintroducing one or more base mismatches into the sequence. Sites ofsiRNA-mRNA complementation are selected which result in optimal mRNAspecificity and maximal mRNA cleavage.

III. RNAi Agents

The present invention includes siRNA molecules designed, for example, asdescribed above. The siRNA molecules of the invention can be chemicallysynthesized, or can be transcribed in vitro from a DNA template, or invivo from e.g., shRNA, or by using recombinant human DICER enzyme, tocleave in vitro transcribed dsRNA templates into pools of 20-, 21- or23-bp duplex RNA mediating RNAi. The siRNA molecules can be designedusing any method known in the art.

In one aspect, instead of the RNAi agent being an interferingribonucleic acid, e.g., an siRNA or shRNA as described above, the RNAiagent can encode an interfering ribonucleic acid, e.g., an shRNA, asdescribed above. In other words, the RNAi agent can be a transcriptionaltemplate of the interfering ribonucleic acid. Thus, RNAi agents of thepresent invention can also include small hairpin RNAs (shRNAs), andexpression constructs engineered to express shRNAs. Transcription ofshRNAs is initiated at a polymerase III (pol III) promoter, and isthought to be terminated at position 2 of a 4-5-thymine transcriptiontermination site. Upon expression, shRNAs are thought to fold into astem-loop structure with 3′ UU-overhangs; subsequently, the ends ofthese shRNAs are processed, converting the shRNAs into siRNA-likemolecules of about 21-23 nucleotides (Brummelkamp et al., 2002; Lee etal., 2002, Supra; Miyagishi et al., 2002; Paddison et al., 2002, supra;Paul et al., 2002, supra; Sui et al., 2002 supra; Yu et al., 2002,supra. More information about shRNA design and use can be found on theinternet at the following addresses:katandin.cshl.org:9331/RNAi/docs/BseRI-BamHI_Strategy.pdf andkatandin.cshl.org:9331/RNAi/docs/Web_version_of_PCR_strategy1.pdf).

Expression constructs of the present invention include any constructsuitable for use in the appropriate expression system and include, butare not limited to, retroviral vectors, linear expression cassettes,plasmids and viral or virally-derived vectors, as known in the art. Suchexpression constructs can include one or more inducible promoters, RNAPol III promoter systems such as U6 snRNA promoters or H1 RNA polymeraseIII promoters, or other promoters known in the art. The constructs caninclude one or both strands of the siRNA. Expression constructsexpressing both strands can also include loop structures linking bothstrands, or each strand can be separately transcribed from separatepromoters within the same construct. Each strand can also be transcribedfrom a separate expression construct. (Tuschl, T., 2002, Supra).

Synthetic siRNAs can be delivered into cells by methods known in theart, including cationic liposome transfection and electroporation. Toobtain longer term suppression of the target genes and to facilitatedelivery under certain circumstances, one or more siRNA can be expressedwithin cells from recombinant DNA constructs. Such methods forexpressing siRNA duplexes within cells from recombinant DNA constructsto allow longer-term target gene suppression in cells are known in theart, including mammalian Pol III promoter systems (e.g., H1 or U6/snRNApromoter systems (Tuschl, T., 2002, supra) capable of expressingfunctional double-stranded siRNAs; (Bagella et al., 1998; Lee et al.,2002, supra; Miyagishi et al., 2002, supra; Paul et al., 2002, supra; Yuet al., 2002, supra; Sui et al., 2002, supra). Transcriptionaltermination by RNA Pol III occurs at runs of four consecutive T residuesin the DNA template, providing a mechanism to end the siRNA transcriptat a specific sequence. The siRNA is complementary to the sequence ofthe target gene in 5′-3′ and 3′-5′ orientations, and the two strands ofthe siRNA can be expressed in the same construct or in separateconstructs. Hairpin siRNAs, driven by H1 or U6 snRNA promoter andexpressed in cells, can inhibit target gene expression (Bagella et al.,1998; Lee et al., 2002, supra; Miyagishi et al., 2002, supra; Paul etal., 2002, supra; Yu et al., 2002), supra; Sui et al., 2002, supra).Constructs containing siRNA sequence under the control of T7 promoteralso make functional siRNAs when co-transfected into the cells with avector expressing T7 RNA polymerase (Jacque et al., 2002, supra). Asingle construct may contain multiple sequences coding for siRNAs, suchas multiple regions of the target gene, targeting the same gene ormultiple genes, and can be driven, for example, by separate PolIIIpromoter sites.

Animal cells express a range of noncoding RNAs of approximately 22nucleotides termed micro RNA (miRNAs) which can regulate gene expressionat the post transcriptional or translational level during animaldevelopment. One common feature of miRNAs is that they are all excisedfrom an approximately 70 nucleotide precursor RNA stem-loop, probably byDicer, an RNase III-type enzyme, or a homolog thereof. By substitutingthe stem sequences of the miRNA precursor with sequence complementary tothe target mRNA, a vector construct that expresses the engineeredprecursor can be used to produce siRNAs to initiate RNAi againstspecific mRNA targets in mammalian cells (Zeng et al., 2002, supra).When expressed by DNA vectors containing polymerase III promoters,micro-RNA designed hairpins can silence gene expression (McManus et al.,2002, supra). MicroRNAs targeting polymorphisms may also be useful forblocking translation of mutant proteins, in the absence ofsiRNA-mediated gene-silencing. Such applications may be useful insituations, for example, where a designed siRNA caused off-targetsilencing of wild type protein.

Viral-mediated delivery mechanisms can also be used to induce specificsilencing of targeted genes through expression of siRNA, for example, bygenerating recombinant adenoviruses harboring siRNA under RNA Pol IIpromoter transcription control (Xia et al., 2002, supra). Infection ofHeLa cells by these recombinant adenoviruses allows for diminishedendogenous target gene expression. Injection of the recombinantadenovirus vectors into transgenic mice expressing the target genes ofthe siRNA results in in vivo reduction of target gene expression. Id. Inan animal model, whole-embryo electroporation can efficiently deliversynthetic siRNA into post-implantation mouse embryos (Calegari et al.,2002). In adult mice, efficient delivery of siRNA can be accomplished by“high-pressure” delivery technique, a rapid injection (within 5 seconds)of a large volume of siRNA containing solution into animal via the tailvein (Liu et al., 1999, supra; McCaffrey et al., 2002, supra; Lewis etal., 2002. Nanoparticles and liposomes can also be used to deliver siRNAinto animals. In certain exemplary embodiments, recombinantadeno-associated viruses (rAAVs) and their associated vectors can beused to deliver one or more siRNAs into cells, e.g., neural cells (e.g.,brain cells) (US Patent Applications 2014/0296486, 2010/0186103,2008/0269149, 2006/0078542 and 2005/0220766).

The nucleic acid compositions of the invention include both unmodifiedsiRNAs and modified siRNAs as known in the art, such as crosslinkedsiRNA derivatives or derivatives having non-nucleotide moieties linked,for example to their 3′ or 5′ ends. Modifying siRNA derivatives in thisway may improve cellular uptake or enhance cellular targeting activitiesof the resulting siRNA derivative as compared to the correspondingsiRNA, are useful for tracing the siRNA derivative in the cell, orimprove the stability of the siRNA derivative compared to thecorresponding siRNA.

Engineered RNA precursors, introduced into cells or whole organisms asdescribed herein, will lead to the production of a desired siRNAmolecule. Such an siRNA molecule will then associate with endogenousprotein components of the RNAi pathway to bind to and target a specificmRNA sequence for cleavage and destruction. In this fashion, the mRNA tobe targeted by the siRNA generated from the engineered RNA precursorwill be depleted from the cell or organism, leading to a decrease in theconcentration of the protein encoded by that mRNA in the cell ororganism. The RNA precursors are typically nucleic acid molecules thatindividually encode either one strand of a dsRNA or encode the entirenucleotide sequence of an RNA hairpin loop structure.

The nucleic acid compositions of the invention can be unconjugated orcan be conjugated to another moiety, such as a nanoparticle, to enhancea property of the compositions, e.g., a pharmacokinetic parameter suchas absorption, efficacy, bioavailability and/or half-life. Theconjugation can be accomplished by methods known in the art, e.g., usingthe methods of Lambert et al., Drug Deliv. Rev.: 47(1), 99-112 (2001)(describes nucleic acids loaded to polyalkylcyanoacrylate(PACA)nanoparticles); Fattal et al., J. Control Release 53(1-3):137-43(1998) (describes nucleic acids bound to nanoparticles); Schwab et al.,Ann. Oncol. Suppl. 4:55-8 (1994) (describes nucleic acids linked tointercalating agents, hydrophobic groups, polycations or PACAnanoparticles); and Godard et al., Eur. J. Biochem. 232(2):404-(1995)(describes nucleic acids linked to nanoparticles).

The nucleic acid molecules of the present invention can also be labeledusing any method known in the art. For instance, the nucleic acidcompositions can be labeled with a fluorophore, e.g., Cy3, fluorescein,or rhodamine. The labeling can be carried out using a kit, e.g., theSILENCER™ siRNA labeling kit (Ambion). Additionally, the siRNA can beradiolabeled, e.g., using ³H, ³²P or other appropriate isotope.

Moreover, because RNAi is believed to progress via at least onesingle-stranded RNA intermediate, the skilled artisan will appreciatethat ss-siRNAs (e.g., the antisense strand of a ds-siRNA) can also bedesigned (e.g., for chemical synthesis) generated (e.g., enzymaticallygenerated) or expressed (e.g., from a vector or plasmid) as describedherein and utilized according to the claimed methodologies. Moreover, ininvertebrates, RNAi can be triggered effectively by long dsRNAs (e.g.,dsRNAs about 100-1000 nucleotides in length, preferably about 200-500,for example, about 250, 300, 350, 400 or 450 nucleotides in length)acting as effectors of RNAi. (Brondani et al., Proc Natl Acad Sci USA.2001 Dec. 4; 98(25):14428-33. Epub 2001 Nov. 27.)

IV. RNA Silencing Agents

In one embodiment, the present invention provides novel RNA silencingagents (e.g., siRNA and shRNAs), methods of making said RNA silencingagents, and methods (e.g., research and/or therapeutic methods) forusing said improved RNA silencing agents (or portions thereof) for RNAsilencing of, for example, an ApoE, C9ORF72, or Htt protein. The RNAsilencing agents comprise an antisense strand (or portions thereof),wherein the antisense strand has sufficient complementary to aheterozygous single nucleotide polymorphism to mediate an RNA-mediatedsilencing mechanism (e.g. RNAi).

In some embodiments, siRNA compounds are provided having one or anycombination of the following properties: (1) fully chemically-stabilized(i.e., no unmodified 2′-OH residues); (2) asymmetry; (3) 11-16 base pairduplexes; (4) alternating pattern of chemically-modified nucleotides(e.g., 2′-fluoro and 2′-methoxy modifications); and (5) single-stranded,fully phosphorothioated tails of 5-8 bases. The number ofphosphorothioate modifications is varied from 6 to 17 total in differentembodiments.

In some embodiments, the siRNA compounds described herein can beconjugated to a variety of targeting agents, including, but not limitedto, cholesterol, DHA, phenyltropanes, cortisol, vitamin A, vitamin D,GaNac, and gangliozides. The cholesterol-modified version showed 5-10fold improvement in efficacy in vitro versus previously used chemicalstabilization patterns (e.g., wherein all purine but not purimidines aremodified) in wide range of cell types (e.g., HeLa, neurons, hepatocytes,trophoblasts).

Certain compounds of the invention having the structural propertiesdescribed above and herein may be referred to as “hsiRNA-ASP”(hydrophobically-modified, small interfering RNA, featuring an advancedstabilization pattern). In addition, this hsiRNA-ASP pattern showed adramatically improved distribution through the brain, spinal cord,delivery to liver, placenta, kidney, spleen and several other tissues,making them accessible for therapeutic intervention.

In liver hsiRNA-ASP delivery specifically to endothelial and kuppercells, but not hepatocytes, making this chemical modification patterncomplimentary rather than competitive technology to GaNac conjugates.

The compounds of the invention can be described in the following aspectsand embodiments.

In a first aspect, provided herein is an oligonucleotide of at least 16contiguous nucleotides, said oligonucleotide having a 5′ end, a 3′ endand complementarity to a target, wherein: (1) the oligonucleotidecomprises alternating 2′-methoxy-ribonucleotides and2′-fluoro-ribonucleotides; (2) the nucleotides at positions 2 and 14from the 5′ end are not 2′-methoxy-ribonucleotides; and (3) thenucleotides are connected via modified linkages as shown in FIG. 1.

a) Design of siRNA Molecules

An siRNA molecule of the invention is a duplex consisting of a sensestrand and complementary antisense strand. Preferably, the siRNAmolecule has a length from about 10-50 or more nucleotides, i.e., eachstrand comprises 10-50 nucleotides (or nucleotide analogs). Morepreferably, the siRNA molecule has a length from about 15-30, e.g., 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30nucleotides in each strand, wherein one of the strands is sufficientlycomplementary to a target region. Preferably, the strands are alignedsuch that there are at least 1, 2, or 3 bases at the end of the strandswhich do not align (i.e., for which no complementary bases occur in theopposing strand) such that an overhang of 1, 2 or 3 residues occurs atone or both ends of the duplex when strands are annealed. Preferably,the siRNA molecule has a length from about 10-50 or more nucleotides,i.e., each strand comprises 10-50 nucleotides (or nucleotide analogs).More preferably, the siRNA molecule has a length from about 15-30, e.g.,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30nucleotides in each strand, wherein one of the strands is substantiallycomplementary to a target sequence, and the other strand is identical orsubstantially identical to the first strand.

In some embodiments, the antisense strand is 20 nucleotides in lengthand the sense strand is 15 nucleotides in length or 16 nucleotides inlength.

In some embodiments, the antisense strand is 21 nucleotides in lengthand the sense strand is 15 nucleotides in length or 16 nucleotides inlength.

In some embodiments, the antisense strand is 20 nucleotides in length or21 nucleotides in length and the sense strand is 15 nucleotides inlength.

In some embodiments, the antisense strand is 20 nucleotides in length or21 nucleotides in length and the sense strand is 16 nucleotides inlength.

In some embodiments, the antisense strand is 20 nucleotides in lengthand the sense strand is 15 nucleotides in length.

In some embodiments, the antisense strand is 21 nucleotides in lengthand the sense strand is 16 nucleotides in length.

Usually, siRNAs can be designed by using any method known in the art,for instance, by using the following protocol:

2. The sense strand of the siRNA is designed based on the sequence ofthe selected target site. Preferably the sense strand includes about 19to 25 nucleotides, e.g., 19, 20, 21, 22, 23, 24 or 25 nucleotides. Morepreferably, the sense strand includes 21, 22 or 23 nucleotides. Theskilled artisan will appreciate, however, that siRNAs having a length ofless than 19 nucleotides or greater than 25 nucleotides can alsofunction to mediate RNAi. Accordingly, siRNAs of such length are alsowithin the scope of the instant invention, provided that they retain theability to mediate RNAi. Longer RNA silencing agents have beendemonstrated to elicit an interferon or Protein Kinase R (PKR) responsein certain mammalian cells which may be undesirable. Preferably, the RNAsilencing agents of the invention do not elicit a PKR response (i.e.,are of a sufficiently short length). However, longer RNA silencingagents may be useful, for example, in cell types incapable of generatinga PKR response or in situations where the PKR response has beendown-regulated or dampened by alternative means.

The siRNA molecules of the invention have sufficient complementaritywith the target sequence such that the siRNA can mediate RNAi. Ingeneral, siRNA containing nucleotide sequences sufficiently identical toa target sequence portion of the target gene to effect RISC-mediatedcleavage of the target gene are preferred. Accordingly, in a preferredembodiment, the sense strand of the siRNA is designed to have a sequencesufficiently identical to a portion of the target. For example, thesense strand may have 100% identity to the target site. However, 100%identity is not required. Greater than 80% identity, e.g., 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or even 100% identity, between the sense strand andthe target RNA sequence is preferred. The invention has the advantage ofbeing able to tolerate certain sequence variations to enhance efficiencyand specificity of RNAi. In one embodiment, the sense strand has 4, 3,2, 1, or 0 mismatched nucleotide(s) with a target region, such as atarget region that differs by at least one base pair between a wild-typeand mutant allele, e.g., a target region comprising the gain-of-functionmutation, and the other strand is identical or substantially identicalto the first strand. Moreover, siRNA sequences with small insertions ordeletions of 1 or 2 nucleotides may also be effective for mediatingRNAi. Alternatively, siRNA sequences with nucleotide analogsubstitutions or insertions can be effective for inhibition.

Sequence identity may be determined by sequence comparison and alignmentalgorithms known in the art. To determine the percent identity of twonucleic acid sequences (or of two amino acid sequences), the sequencesare aligned for optimal comparison purposes (e.g., gaps can beintroduced in the first sequence or second sequence for optimalalignment). The nucleotides (or amino acid residues) at correspondingnucleotide (or amino acid) positions are then compared. When a positionin the first sequence is occupied by the same residue as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., % homology=number of identical positions/totalnumber of positions×100), optionally penalizing the score for the numberof gaps introduced and/or length of gaps introduced.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In one embodiment, the alignment generated over a certainportion of the sequence aligned having sufficient identity but not overportions having low degree of identity (i.e., a local alignment). Apreferred, non-limiting example of a local alignment algorithm utilizedfor the comparison of sequences is the algorithm of Karlin and Altschul(1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as in Karlin andAltschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithmis incorporated into the BLAST programs (version 2.0) of Altschul, etal. (1990) J. Mol. Biol. 215:403-10.

In another embodiment, the alignment is optimized by introducingappropriate gaps and percent identity is determined over the length ofthe aligned sequences (i.e., a gapped alignment). To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. In another embodiment, the alignment is optimized byintroducing appropriate gaps and percent identity is determined over theentire length of the sequences aligned (i.e., a global alignment). Apreferred, non-limiting example of a mathematical algorithm utilized forthe global comparison of sequences is the algorithm of Myers and Miller,CABIOS (1989). Such an algorithm is incorporated into the ALIGN program(version 2.0) which is part of the GCG sequence alignment softwarepackage. When utilizing the ALIGN program for comparing amino acidsequences, a PAM120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used.

3. The antisense or guide strand of the siRNA is routinely the samelength as the sense strand and includes complementary nucleotides. Inone embodiment, the guide and sense strands are fully complementary,i.e., the strands are blunt-ended when aligned or annealed. In anotherembodiment, the strands of the siRNA can be paired in such a way as tohave a 3′ overhang of 1 to 7 (e.g., 2, 3, 4, 5, 6 or 7), or 1 to 4,e.g., 2, 3 or 4 nucleotides. Overhangs can comprise (or consist of)nucleotides corresponding to the target gene sequence (or complementthereof). Alternatively, overhangs can comprise (or consist of)deoxyribonucleotides, for example dTs, or nucleotide analogs, or othersuitable non-nucleotide material. Thus, in another embodiment, thenucleic acid molecules may have a 3′ overhang of 2 nucleotides, such asTT. The overhanging nucleotides may be either RNA or DNA. As notedabove, it is desirable to choose a target region wherein the mutant:wildtype mismatch is a purine:purine mismatch.

4. Using any method known in the art, compare the potential targets tothe appropriate genome database (human, mouse, rat, etc.) and eliminatefrom consideration any target sequences with significant homology toother coding sequences. One such method for such sequence homologysearches is known as BLAST, which is available at National Center forBiotechnology Information website.

5. Select one or more sequences that meet your criteria for evaluation.

Further general information about the design and use of siRNA may befound in “The siRNA User Guide,” available at The Max-Plank-Institut furBiophysikalische Chemie website.

Alternatively, the siRNA may be defined functionally as a nucleotidesequence (or oligonucleotide sequence) that is capable of hybridizingwith the target sequence (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mMEDTA, 50° C. or 70° C. hybridization for 12-16 hours; followed bywashing). Additional preferred hybridization conditions includehybridization at 70° C. in 1×SSC or 50° C. in 1×SSC, 50% formamidefollowed by washing at 70° C. in 0.3×SSC or hybridization at 70° C. in4×SSC or 50° C. in 4×SSC, 50% formamide followed by washing at 67° C. in1×SSC. The hybridization temperature for hybrids anticipated to be lessthan 50 base pairs in length should be 5-10° C. less than the meltingtemperature (Tm) of the hybrid, where Tm is determined according to thefollowing equations. For hybrids less than 18 base pairs in length, Tm(°C.)=2(# of A+T bases)+4(# of G+C bases). For hybrids between 18 and 49base pairs in length, Tm(° C.)=81.5+16.6(log 10[Na⁺])+0.41(%G+C)−(600/N), where N is the number of bases in the hybrid, and [Na⁺] isthe concentration of sodium ions in the hybridization buffer ([Na⁺] for1×SSC=0.165 M). Additional examples of stringency conditions forpolynucleotide hybridization are provided in Sambrook, J., E. F.Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters9 and 11, and Current Protocols in Molecular Biology, 1995, F. M.Ausubel et al., eds., John Wiley & Sons, Inc., sections 2.10 and6.3-6.4, incorporated herein by reference.

Negative control siRNAs should have the same nucleotide composition asthe selected siRNA, but without significant sequence complementarity tothe appropriate genome. Such negative controls may be designed byrandomly scrambling the nucleotide sequence of the selected siRNA. Ahomology search can be performed to ensure that the negative controllacks homology to any other gene in the appropriate genome. In addition,negative control siRNAs can be designed by introducing one or more basemismatches into the sequence.

6. To validate the effectiveness by which siRNAs destroy target mRNAs(e.g., wild-type or mutant mRNA), the siRNA may be incubated with targetcDNA in a Drosophila-based in vitro mRNA expression system. Radiolabeledwith ³²P, newly synthesized target mRNAs are detectedautoradiographically on an agarose gel. The presence of cleaved targetmRNA indicates mRNA nuclease activity. Suitable controls includeomission of siRNA and use of non-target cDNA. Alternatively, controlsiRNAs are selected having the same nucleotide composition as theselected siRNA, but without significant sequence complementarity to theappropriate target gene. Such negative controls can be designed byrandomly scrambling the nucleotide sequence of the selected siRNA. Ahomology search can be performed to ensure that the negative controllacks homology to any other gene in the appropriate genome. In addition,negative control siRNAs can be designed by introducing one or more basemismatches into the sequence.

siRNAs may be designed to target any of the target sequences describedsupra. Said siRNAs comprise an antisense strand which is sufficientlycomplementary with the target sequence to mediate silencing of thetarget sequence. In some embodiments, the RNA silencing agent is asiRNA.

In some embodiments, the siRNA comprises a sense strand comprising alinkage set forth at FIG. 1, or an antisense strand comprising a linkageset forth at FIG. 1.

Sites of siRNA-mRNA complementation are selected which result in optimalmRNA specificity and maximal mRNA cleavage.

b) siRNA-Like Molecules

siRNA-like molecules of the invention have a sequence (i.e., have astrand having a sequence) that is “sufficiently complementary” to atarget sequence of an mRNA to direct gene silencing either by RNAi ortranslational repression. siRNA-like molecules are designed in the sameway as siRNA molecules, but the degree of sequence identity between thesense strand and target RNA approximates that observed between an miRNAand its target. In general, as the degree of sequence identity between amiRNA sequence and the corresponding target gene sequence is decreased,the tendency to mediate post-transcriptional gene silencing bytranslational repression rather than RNAi is increased. Therefore, in analternative embodiment, where post-transcriptional gene silencing bytranslational repression of the target gene is desired, the miRNAsequence has partial complementarity with the target gene sequence. Insome embodiments, the miRNA sequence has partial complementarity withone or more short sequences (complementarity sites) dispersed within thetarget mRNA (e.g. within the 3′-UTR of the target mRNA) (Hutvagner andZamore, Science, 2002; Zeng et al., Mol. Cell, 2002; Zeng et al., RNA,2003; Doench et al., Genes & Dev., 2003). Since the mechanism oftranslational repression is cooperative, multiple complementarity sites(e.g., 2, 3, 4, 5, or 6) may be targeted in some embodiments.

The capacity of a siRNA-like duplex to mediate RNAi or translationalrepression may be predicted by the distribution of non-identicalnucleotides between the target gene sequence and the nucleotide sequenceof the silencing agent at the site of complementarity. In oneembodiment, where gene silencing by translational repression is desired,at least one non-identical nucleotide is present in the central portionof the complementarity site so that duplex formed by the miRNA guidestrand and the target mRNA contains a central “bulge” (Doench J G etal., Genes & Dev., 2003). In another embodiment 2, 3, 4, 5, or 6contiguous or non-contiguous non-identical nucleotides are introduced.The non-identical nucleotide may be selected such that it forms a wobblebase pair (e.g., G:U) or a mismatched base pair (G:A, C:A, C:U, G:G,A:A, C:C, U:U). In a further preferred embodiment, the “bulge” iscentered at nucleotide positions 12 and 13 from the 5′ end of the miRNAmolecule.

c) Short Hairpin RNA (shRNA) Molecules

In certain featured embodiments, the instant invention provides shRNAscapable of mediating RNA silencing of a target sequence with enhancedselectivity. In contrast to siRNAs, shRNAs mimic the natural precursorsof micro RNAs (miRNAs) and enter at the top of the gene silencingpathway. For this reason, shRNAs are believed to mediate gene silencingmore efficiently by being fed through the entire natural gene silencingpathway.

miRNAs are noncoding RNAs of approximately 22 nucleotides which canregulate gene expression at the post transcriptional or translationallevel during plant and animal development. One common feature of miRNAsis that they are all excised from an approximately 70 nucleotideprecursor RNA stem-loop termed pre-miRNA, probably by Dicer, an RNaseIII-type enzyme, or a homolog thereof. Naturally-occurring miRNAprecursors (pre-miRNA) have a single strand that forms a duplex stemincluding two portions that are generally complementary, and a loop,that connects the two portions of the stem. In typical pre-miRNAs, thestem includes one or more bulges, e.g., extra nucleotides that create asingle nucleotide “loop” in one portion of the stem, and/or one or moreunpaired nucleotides that create a gap in the hybridization of the twoportions of the stem to each other. Short hairpin RNAs, or engineeredRNA precursors, of the invention are artificial constructs based onthese naturally occurring pre-miRNAs, but which are engineered todeliver desired RNA silencing agents (e.g., siRNAs of the invention). Bysubstituting the stem sequences of the pre-miRNA with sequencecomplementary to the target mRNA, a shRNA is formed. The shRNA isprocessed by the entire gene silencing pathway of the cell, therebyefficiently mediating RNAi.

The requisite elements of a shRNA molecule include a first portion and asecond portion, having sufficient complementarity to anneal or hybridizeto form a duplex or double-stranded stem portion. The two portions neednot be fully or perfectly complementary. The first and second “stem”portions are connected by a portion having a sequence that hasinsufficient sequence complementarity to anneal or hybridize to otherportions of the shRNA. This latter portion is referred to as a “loop”portion in the shRNA molecule. The shRNA molecules are processed togenerate siRNAs. shRNAs can also include one or more bulges, i.e., extranucleotides that create a small nucleotide “loop” in a portion of thestem, for example a one-, two- or three-nucleotide loop. The stemportions can be the same length, or one portion can include an overhangof, for example, 1-5 nucleotides. The overhanging nucleotides caninclude, for example, uracils (Us), e.g., all Us. Such Us are notablyencoded by thymidines (Ts) in the shRNA-encoding DNA which signal thetermination of transcription.

In shRNAs (or engineered precursor RNAs) of the instant invention, oneportion of the duplex stem is a nucleic acid sequence that iscomplementary (or anti-sense) to the target sequence. Preferably, onestrand of the stem portion of the shRNA is sufficiently complementary(e.g., antisense) to a target RNA (e.g., mRNA) sequence to mediatedegradation or cleavage of said target RNA via RNA interference (RNAi).Thus, engineered RNA precursors include a duplex stem with two portionsand a loop connecting the two stem portions. The antisense portion canbe on the 5′ or 3′ end of the stem. The stem portions of a shRNA arepreferably about 15 to about 50 nucleotides in length. Preferably thetwo stem portions are about 18 or 19 to about 21, 22, 23, 24, 25, 30,35, 37, 38, 39, or 40 or more nucleotides in length. In preferredembodiments, the length of the stem portions should be 21 nucleotides orgreater. When used in mammalian cells, the length of the stem portionsshould be less than about 30 nucleotides to avoid provoking non-specificresponses like the interferon pathway. In non-mammalian cells, the stemcan be longer than 30 nucleotides. In fact, the stem can include muchlarger sections complementary to the target mRNA (up to, and includingthe entire mRNA). In fact, a stem portion can include much largersections complementary to the target mRNA (up to, and including theentire mRNA).

The two portions of the duplex stem must be sufficiently complementaryto hybridize to form the duplex stem. Thus, the two portions can be, butneed not be, fully or perfectly complementary. In addition, the two stemportions can be the same length, or one portion can include an overhangof 1, 2, 3, or 4 nucleotides. The overhanging nucleotides can include,for example, uracils (Us), e.g., all Us. The loop in the shRNAs orengineered RNA precursors may differ from natural pre-miRNA sequences bymodifying the loop sequence to increase or decrease the number of pairednucleotides, or replacing all or part of the loop sequence with atetraloop or other loop sequences. Thus, the loop in the shRNAs orengineered RNA precursors can be 2, 3, 4, 5, 6, 7, 8, 9, or more, e.g.,15 or 20, or more nucleotides in length.

The loop in the shRNAs or engineered RNA precursors may differ fromnatural pre-miRNA sequences by modifying the loop sequence to increaseor decrease the number of paired nucleotides, or replacing all or partof the loop sequence with a tetraloop or other loop sequences. Thus, theloop portion in the shRNA can be about 2 to about 20 nucleotides inlength, i.e., about 2, 3, 4, 5, 6, 7, 8, 9, or more, e.g., 15 or 20, ormore nucleotides in length. A preferred loop consists of or comprises a“tetraloop” sequences. Exemplary tetraloop sequences include, but arenot limited to, the sequences GNRA, where N is any nucleotide and R is apurine nucleotide, GGGG, and UUUU.

In some embodiments, shRNAs of the invention include the sequences of adesired siRNA molecule described supra. In other embodiments, thesequence of the antisense portion of a shRNA can be designed essentiallyas described above or generally by selecting an 18, 19, 20, 21nucleotides, or longer, sequence from within the target RNA, forexample, from a region 100 to 200 or 300 nucleotides upstream ordownstream of the start of translation. In general, the sequence can beselected from any portion of the target RNA (e.g., mRNA) including the5′ UTR (untranslated region), coding sequence, or 3′ UTR. This sequencecan optionally follow immediately after a region of the target genecontaining two adjacent AA nucleotides. The last two nucleotides of thenucleotide sequence can be selected to be UU. This 21 or so nucleotidesequence is used to create one portion of a duplex stem in the shRNA.This sequence can replace a stem portion of a wild-type pre-miRNAsequence, e.g., enzymatically, or is included in a complete sequencethat is synthesized. For example, one can synthesize DNAoligonucleotides that encode the entire stem-loop engineered RNAprecursor, or that encode just the portion to be inserted into theduplex stem of the precursor, and using restriction enzymes to build theengineered RNA precursor construct, e.g., from a wild-type pre-miRNA.

Engineered RNA precursors include in the duplex stem the 21-22 or sonucleotide sequences of the siRNA or siRNA-like duplex desired to beproduced in vivo. Thus, the stem portion of the engineered RNA precursorincludes at least 18 or 19 nucleotide pairs corresponding to thesequence of an exonic portion of the gene whose expression is to bereduced or inhibited. The two 3′ nucleotides flanking this region of thestem are chosen so as to maximize the production of the siRNA from theengineered RNA precursor and to maximize the efficacy of the resultingsiRNA in targeting the corresponding mRNA for translational repressionor destruction by RNAi in vivo and in vitro.

In some embodiments, shRNAs of the invention include miRNA sequences,optionally end-modified miRNA sequences, to enhance entry into RISC. ThemiRNA sequence can be similar or identical to that of any naturallyoccurring miRNA (see e.g. The miRNA Registry; Griffiths-Jones S, Nuc.Acids Res., 2004). Over one thousand natural miRNAs have been identifiedto date and together they are thought to comprise about 1% of allpredicted genes in the genome. Many natural miRNAs are clusteredtogether in the introns of pre-mRNAs and can be identified in silicousing homology-based searches (Pasquinelli et al., 2000; Lagos-Quintanaet al., 2001; Lau et al., 2001; Lee and Ambros, 2001) or computeralgorithms (e.g. MiRScan, MiRSeeker) that predict the capability of acandidate miRNA gene to form the stem loop structure of a pri-mRNA (Gradet al., Mol. Cell., 2003; Lim et al., Genes Dev., 2003; Lim et al.,Science, 2003; Lai E C et al., Genome Bio., 2003). An online registryprovides a searchable database of all published miRNA sequences (ThemiRNA Registry at the Sanger Institute website; Griffiths-Jones S, Nuc.Acids Res., 2004). Exemplary, natural miRNAs include lin-4, let-7,miR-10, mirR-15, miR-16, miR-168, miR-175, miR-196 and their homologs,as well as other natural miRNAs from humans and certain model organismsincluding Drosophila melanogaster, Caenorhabditis elegans, zebrafish,Arabidopsis thalania, Mus musculus, and Rattus norvegicus as describedin International PCT Publication No. WO 03/029459.

Naturally-occurring miRNAs are expressed by endogenous genes in vivo andare processed from a hairpin or stem-loop precursor (pre-miRNA orpri-miRNAs) by Dicer or other RNAses (Lagos-Quintana et al., Science,2001; Lau et al., Science, 2001; Lee and Ambros, Science, 2001;Lagos-Quintana et al., Curr. Biol., 2002; Mourelatos et al., Genes Dev.,2002; Reinhart et al., Science, 2002; Ambros et al., Curr. Biol., 2003;Brennecke et al., 2003; Lagos-Quintana et al., RNA, 2003; Lim et al.,Genes Dev., 2003; Lim et al., Science, 2003). miRNAs can existtransiently in vivo as a double-stranded duplex, but only one strand istaken up by the RISC complex to direct gene silencing. Certain miRNAs,e.g., plant miRNAs, have perfect or near-perfect complementarity totheir target mRNAs and, hence, direct cleavage of the target mRNAs.Other miRNAs have less than perfect complementarity to their targetmRNAs and, hence, direct translational repression of the target mRNAs.The degree of complementarity between an miRNA and its target mRNA isbelieved to determine its mechanism of action. For example, perfect ornear-perfect complementarity between a miRNA and its target mRNA ispredictive of a cleavage mechanism (Yekta et al., Science, 2004),whereas less than perfect complementarity is predictive of atranslational repression mechanism. In particular embodiments, the miRNAsequence is that of a naturally-occurring miRNA sequence, the aberrantexpression or activity of which is correlated with an miRNA disorder.

d) Dual Functional Oligonucleotide Tethers

In other embodiments, the RNA silencing agents of the present inventioninclude dual functional oligonucleotide tethers useful for theintercellular recruitment of a miRNA. Animal cells express a range ofmiRNAs, noncoding RNAs of approximately 22 nucleotides which canregulate gene expression at the post transcriptional or translationallevel. By binding a miRNA bound to RISC and recruiting it to a targetmRNA, a dual functional oligonucleotide tether can repress theexpression of genes involved e.g., in the arteriosclerotic process. Theuse of oligonucleotide tethers offers several advantages over existingtechniques to repress the expression of a particular gene. First, themethods described herein allow an endogenous molecule (often present inabundance), an miRNA, to mediate RNA silencing. Accordingly, the methodsdescribed herein obviate the need to introduce foreign molecules (e.g.,siRNAs) to mediate RNA silencing. Second, the RNA-silencing agents and,in particular, the linking moiety (e.g., oligonucleotides such as the2′-O-methyl oligonucleotide), can be made stable and resistant tonuclease activity. As a result, the tethers of the present invention canbe designed for direct delivery, obviating the need for indirectdelivery (e.g. viral) of a precursor molecule or plasmid designed tomake the desired agent within the cell. Third, tethers and theirrespective moieties, can be designed to conform to specific mRNA sitesand specific miRNAs. The designs can be cell and gene product specific.Fourth, the methods disclosed herein leave the mRNA intact, allowing oneskilled in the art to block protein synthesis in short pulses using thecell's own machinery. As a result, these methods of RNA silencing arehighly regulatable.

The dual functional oligonucleotide tethers (“tethers”) of the inventionare designed such that they recruit miRNAs (e.g., endogenous cellularmiRNAs) to a target mRNA so as to induce the modulation of a gene ofinterest. In preferred embodiments, the tethers have the formula T-L-μ,wherein T is an mRNA targeting moiety, L is a linking moiety, and is anmiRNA recruiting moiety. Any one or more moiety may be double stranded.Preferably, however, each moiety is single stranded.

Moieties within the tethers can be arranged or linked (in the 5′ to 3′direction) as depicted in the formula T-L-μ (i.e., the 3′ end of thetargeting moiety linked to the 5′ end of the linking moiety and the 3′end of the linking moiety linked to the 5′ end of the miRNA recruitingmoiety). Alternatively, the moieties can be arranged or linked in thetether as follows: μ-T-L (i.e., the 3′ end of the miRNA recruitingmoiety linked to the 5′ end of the linking moiety and the 3′ end of thelinking moiety linked to the 5′ end of the targeting moiety).

The mRNA targeting moiety, as described above, is capable of capturing aspecific target mRNA. According to the invention, expression of thetarget mRNA is undesirable, and, thus, translational repression of themRNA is desired. The mRNA targeting moiety should be of sufficient sizeto effectively bind the target mRNA. The length of the targeting moietywill vary greatly depending, in part, on the length of the target mRNAand the degree of complementarity between the target mRNA and thetargeting moiety. In various embodiments, the targeting moiety is lessthan about 200, 100, 50, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,10, 9, 8, 7, 6, or 5 nucleotides in length. In a particular embodiment,the targeting moiety is about 15 to about 25 nucleotides in length.

The miRNA recruiting moiety, as described above, is capable ofassociating with a miRNA. According to the invention, the miRNA may beany miRNA capable of repressing the target mRNA. Mammals are reported tohave over 250 endogenous miRNAs (Lagos-Quintana et al. (2002) CurrentBiol. 12:735-739; Lagos-Quintana et al. (2001) Science 294:858-862; andLim et al. (2003) Science 299:1540). In various embodiments, the miRNAmay be any art-recognized miRNA.

The linking moiety is any agent capable of linking the targetingmoieties such that the activity of the targeting moieties is maintained.Linking moieties are preferably oligonucleotide moieties comprising asufficient number of nucleotides such that the targeting agents cansufficiently interact with their respective targets. Linking moietieshave little or no sequence homology with cellular mRNA or miRNAsequences. Exemplary linking moieties include one or more2′-O-methylnucleotides, e.g., 2′-p-methyladenosine,2′-O-methylthymidine, 2′-O-methylguanosine or 2′-O-methyluridine.

e) Gene Silencing Oligonucleotides

In certain exemplary embodiments, gene expression (e.g., target geneexpression) can be modulated using oligonucleotide-based compoundscomprising two or more single stranded antisense oligonucleotides thatare linked through their 5′-ends that allow the presence of two or moreaccessible 3′-ends to effectively inhibit or decrease target geneexpression. Such linked oligonucleotides are also known as GeneSilencing Oligonucleotides (GSOs). (See, e.g., U.S. Pat. No. 8,431,544assigned to Idera Pharmaceuticals, Inc., incorporated herein byreference in its entirety for all purposes.) Provided herein are noveland improved GSOs comprising intersubunit linkages according to Formula(I) and its embodiments.

The linkage at the 5′ ends of the GSOs is independent of the otheroligonucleotide linkages and may be directly via 5′, 3′ or 2′ hydroxylgroups, or indirectly, via a non-nucleotide linker or a nucleoside,utilizing either the 2′ or 3′ hydroxyl positions of the nucleoside.Linkages may also utilize a functionalized sugar or nucleobase of a 5′terminal nucleotide.

GSOs can comprise two identical or different sequences conjugated attheir 5′-5′ ends via a phosphodiester, phosphorothioate ornon-nucleoside linker. Such compounds may comprise 15 to 27 nucleotidesthat are complementary to specific portions of mRNA targets of interestfor antisense down regulation of gene product. GSOs that compriseidentical sequences can bind to a specific mRNA via Watson-Crickhydrogen bonding interactions and inhibit protein expression. GSOs thatcomprise different sequences are able to bind to two or more differentregions of one or more mRNA target and inhibit protein expression. Suchcompounds are comprised of heteronucleotide sequences complementary totarget mRNA and form stable duplex structures through Watson-Crickhydrogen bonding. Under certain conditions, GSOs containing two free3′-ends (5′-5′-attached antisense) can be more potent inhibitors of geneexpression than those containing a single free 3′-end or no free 3′-end.

In some embodiments, the non-nucleotide linker is glycerol or a glycerolhomolog of the formula HO—(CH₂)_(o)—CH(OH)—(CH₂)_(p)—OH, wherein o and pindependently are integers from 1 to about 6, from 1 to about 4 or from1 to about 3. In some other embodiments, the non-nucleotide linker is aderivative of 1,3-diamino-2-hydroxypropane. Some such derivatives havethe formula HO—(CH₂)_(m)—C(O)NH—CH₂—CH(OH)—CH₂—NHC(O)—(CH₂)_(m)—OH,wherein m is an integer from 0 to about 10, from 0 to about 6, from 2 toabout 6 or from 2 to about 4.

Some non-nucleotide linkers permit attachment of more than two GSOcomponents. For example, the non-nucleotide linker glycerol has threehydroxyl groups to which GSO components may be covalently attached. Someoligonucleotide-based compounds of the invention, therefore, comprisetwo or more oligonucleotides linked to a nucleotide or a non-nucleotidelinker. Such oligonucleotides according to the invention are referred toas being “branched.”

In some embodiments, GSOs are at least 14 nucleotides in length. Incertain exemplary embodiments, GSOs are 15 to 40 nucleotides long or 20to 30 nucleotides in length. Thus, the component oligonucleotides ofGSOs can independently be 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40nucleotides in length.

These oligonucleotides can be prepared by the art recognized methodssuch as phosphoramidate or H-phosphonate chemistry which can be carriedout manually or by an automated synthesizer. These oligonucleotides mayalso be modified in a number of ways without compromising their abilityto hybridize to mRNA. Such modifications may include at least oneinternucleotide linkage of the oligonucleotide being analkylphosphonate, phosphorothioate, phosphorodithioate,methylphosphonate, phosphate ester, alkylphosphonothioate,phosphoramidate, carbamate, carbonate, phosphate hydroxyl, acetamidateor carboxymethyl ester or a combination of these and otherinternucleotide linkages between the 5′ end of one nucleotide and the 3′end of another nucleotide in which the 5′ nucleotide phosphodiesterlinkage has been replaced with any number of chemical groups.

V. Modified RNA Silencing Agents

In certain aspects of the invention, the oligonucleotides, siRNA, andRNA silencing agents (or any portion thereof) of the invention asdescribed supra may be modified such that the activity of the agent isfurther improved. For example, the RNA silencing agents described inSection II supra may be modified with any of the modifications describedinfra. The modifications can, in part, serve to further enhance targetdiscrimination, to enhance stability of the agent (e.g., to preventdegradation), to promote cellular uptake, to enhance the targetefficiency, to improve efficacy in binding (e.g., to the targets), toimprove patient tolerance to the agent, and/or to reduce toxicity.

1) Modifications to Enhance Target Discrimination

In some embodiments, the oligonucleotides, siRNA, and RNA silencingagents of the invention may be substituted with a destabilizingnucleotide to enhance single nucleotide target discrimination (see U.S.application Ser. No. 11/698,689, filed Jan. 25, 2007 and U.S.Provisional Application No. 60/762,225 filed Jan. 25, 2006, both ofwhich are incorporated herein by reference). Such a modification may besufficient to abolish the specificity of the RNA silencing agent for anon-target mRNA (e.g. wild-type mRNA), without appreciably affecting thespecificity of the RNA silencing agent for a target mRNA (e.g.gain-of-function mutant mRNA).

In preferred embodiments, the RNA silencing agents of the invention aremodified by the introduction of at least one universal nucleotide in theantisense strand thereof. Universal nucleotides comprise base portionsthat are capable of base pairing indiscriminately with any of the fourconventional nucleotide bases (e.g. A, G, C, U). A universal nucleotideis preferred because it has relatively minor effect on the stability ofthe RNA duplex or the duplex formed by the guide strand of the RNAsilencing agent and the target mRNA. Exemplary universal nucleotideinclude those having an inosine base portion or an inosine analog baseportion selected from the group consisting of deoxyinosine (e.g.2′-deoxyinosine), 7-deaza-2′-deoxyinosine, 2′-aza-2′-deoxyinosine,PNA-inosine, morpholino-inosine, LNA-inosine, phosphoramidate-inosine,2′-O-methoxyethyl-inosine, and 2′-OMe-inosine. In particularly preferredembodiments, the universal nucleotide is an inosine residue or anaturally occurring analog thereof.

In some embodiments, the RNA silencing agents of the invention aremodified by the introduction of at least one destabilizing nucleotidewithin 5 nucleotides from a specificity-determining nucleotide (i.e.,the nucleotide which recognizes the disease-related polymorphism). Forexample, the destabilizing nucleotide may be introduced at a positionthat is within 5, 4, 3, 2, or 1 nucleotide(s) from aspecificity-determining nucleotide. In exemplary embodiments, thedestabilizing nucleotide is introduced at a position which is 3nucleotides from the specificity-determining nucleotide (i.e., such thatthere are 2 stabilizing nucleotides between the destabilizing nucleotideand the specificity-determining nucleotide). In RNA silencing agentshaving two strands or strand portions (e.g. siRNAs and shRNAs), thedestabilizing nucleotide may be introduced in the strand or strandportion that does not contain the specificity-determining nucleotide. Inpreferred embodiments, the destabilizing nucleotide is introduced in thesame strand or strand portion that contains the specificity-determiningnucleotide.

2) Modifications to Enhance Efficacy and Specificity

In some embodiments, the oligonucleotides, siRNA, and RNA silencingagents of the invention may be altered to facilitate enhanced efficacyand specificity in mediating RNAi according to asymmetry design rules(see U.S. Pat. Nos. 8,309,704, 7,750,144, 8,304,530, 8,329,892 and8,309,705). Such alterations facilitate entry of the antisense strand ofthe siRNA (e.g., a siRNA designed using the methods of the invention oran siRNA produced from a shRNA) into RISC in favor of the sense strand,such that the antisense strand preferentially guides cleavage ortranslational repression of a target mRNA, and thus increasing orimproving the efficiency of target cleavage and silencing. Preferablythe asymmetry of an RNA silencing agent is enhanced by lessening thebase pair strength between the antisense strand 5′ end (AS 5′) and thesense strand 3′ end (S 3′) of the RNA silencing agent relative to thebond strength or base pair strength between the antisense strand 3′ end(AS 3′) and the sense strand 5′ end (S ′5) of said RNA silencing agent.

In one embodiment, the asymmetry of an RNA silencing agent of theinvention may be enhanced such that there are fewer G:C base pairsbetween the 5′ end of the first or antisense strand and the 3′ end ofthe sense strand portion than between the 3′ end of the first orantisense strand and the 5′ end of the sense strand portion. In anotherembodiment, the asymmetry of an RNA silencing agent of the invention maybe enhanced such that there is at least one mismatched base pair betweenthe 5′ end of the first or antisense strand and the 3′ end of the sensestrand portion. Preferably, the mismatched base pair is selected fromthe group consisting of G:A, CA, C:U, G:G, A:A, C:C and U:U. In anotherembodiment, the asymmetry of an RNA silencing agent of the invention maybe enhanced such that there is at least one wobble base pair, e.g., G:U,between the 5′ end of the first or antisense strand and the 3′ end ofthe sense strand portion. In another embodiment, the asymmetry of an RNAsilencing agent of the invention may be enhanced such that there is atleast one base pair comprising a rare nucleotide, e.g., inosine (I).Preferably, the base pair is selected from the group consisting of anI:A, I:U and I:C. In yet another embodiment, the asymmetry of an RNAsilencing agent of the invention may be enhanced such that there is atleast one base pair comprising a modified nucleotide. In preferredembodiments, the modified nucleotide is selected from the groupconsisting of 2-amino-G, 2-amino-A, 2,6-diamino-G, and 2,6-diamino-A.

3) RNA Silencing Agents with Enhanced Stability

The RNA silencing agents of the present invention can be modified toimprove stability in serum or in growth medium for cell cultures. Inorder to enhance the stability, the 3′-residues may be stabilizedagainst degradation, e.g., they may be selected such that they consistof purine nucleotides, particularly adenosine or guanosine nucleotides.Alternatively, substitution of pyrimidine nucleotides by modifiedanalogues, e.g., substitution of uridine by 2′-deoxythymidine istolerated and does not affect the efficiency of RNA interference.

In a one aspect, the invention features RNA silencing agents thatinclude first and second strands wherein the second strand and/or firststrand is modified by the substitution of internal nucleotides withmodified nucleotides, such that in vivo stability is enhanced ascompared to a corresponding unmodified RNA silencing agent. As definedherein, an “internal” nucleotide is one occurring at any position otherthan the 5′ end or 3′ end of nucleic acid molecule, polynucleotide oroligonucleotide. An internal nucleotide can be within a single-strandedmolecule or within a strand of a duplex or double-stranded molecule. Inone embodiment, the sense strand and/or antisense strand is modified bythe substitution of at least one internal nucleotide. In anotherembodiment, the sense strand and/or antisense strand is modified by thesubstitution of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more internal nucleotides. Inanother embodiment, the sense strand and/or antisense strand is modifiedby the substitution of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of theinternal nucleotides. In yet another embodiment, the sense strand and/orantisense strand is modified by the substitution of all of the internalnucleotides.

In one aspect, the invention features RNA silencing agents that are atleast 80% chemically modified. In a preferred embodiment of the presentinvention, the RNA silencing agents may be fully chemically modified,i.e., 100% of the nucleotides are chemically modified.

In a preferred embodiment of the present invention, the RNA silencingagents may contain at least one modified nucleotide analogue. Thenucleotide analogues may be located at positions where thetarget-specific silencing activity, e.g., the RNAi mediating activity ortranslational repression activity is not substantially effected, e.g.,in a region at the 5′-end and/or the 3′-end of the siRNA molecule.Particularly, the ends may be stabilized by incorporating modifiednucleotide analogues.

Exemplary nucleotide analogues include sugar- and/or backbone-modifiedribonucleotides (i.e., include modifications to the phosphate-sugarbackbone). For example, the phosphodiester linkages of natural RNA maybe modified to include at least one of a nitrogen or sulfur heteroatom.In exemplary backbone-modified ribonucleotides, the phosphoester groupconnecting to adjacent ribonucleotides is replaced by a modified group,e.g., of phosphothioate group. In exemplary sugar-modifiedribonucleotides, the 2′ OH-group is replaced by a group selected from H,OR, R, halo, SH, SR, NH₂, NHR, NR₂ or ON, wherein R is C₁-C₆ alkyl,alkenyl or alkynyl and halo is F, Cl, Br or I.

In particular embodiments, the modifications are 2′-fluoro, 2′-aminoand/or 2′-thio modifications. Particularly preferred modificationsinclude 2′-fluoro-cytidine, 2′-fluoro-uridine, 2′-fluoro-adenosine,2′-fluoro-guanosine, 2′-amino-cytidine, 2′-amino-uridine,2′-amino-adenosine, 2-amino-guanosine, 2,6-diaminopurine,4-thio-uridine, and/or 5-amino-allyl-uridine. In a particularembodiment, the 2′-fluoro ribonucleotides are every uridine andcytidine. Additional exemplary modifications include 5-bromo-uridine,5-iodo-uridine, 5-methyl-cytidine, ribo-thymidine, 2-aminopurine,2′-amino-butyryl-pyrene-uridine, 5-fluoro-cytidine, and5-fluoro-uridine. 2′-deoxy-nucleotides and 2′-Ome nucleotides can alsobe used within modified RNA-silencing agents moieties of the instantinvention. Additional modified residues include, deoxy-abasic, inosine,N3-methyl-uridine, N6,N6-dimethyl-adenosine, pseudouridine, purineribonucleoside and ribavirin. In a particularly preferred embodiment,the 2′ moiety is a methyl group such that the linking moiety is a2′-O-methyl oligonucleotide.

In an exemplary embodiment, the RNA silencing agent of the inventioncomprises Locked Nucleic Acids (LNAs). LNAs comprise sugar-modifiednucleotides that resist nuclease activities (are highly stable) andpossess single nucleotide discrimination for mRNA (Elmen et al., NucleicAcids Res., (2005), 33(1): 439-447; Braasch et al. (2003) Biochemistry42:7967-7975, Petersen et al. (2003) Trends Biotechnol 21:74-81). Thesemolecules have 2′-0,4′-C-ethylene-bridged nucleic acids, with possiblemodifications such as 2′-deoxy-2″-fluorouridine. Moreover, LNAs increasethe specificity of oligonucleotides by constraining the sugar moietyinto the 3′-endo conformation, thereby pre-organizing the nucleotide forbase pairing and increasing the melting temperature of theoligonucleotide by as much as 10° C. per base.

In another exemplary embodiment, the RNA silencing agent of theinvention comprises Peptide Nucleic Acids (PNAs). PNAs comprise modifiednucleotides in which the sugar-phosphate portion of the nucleotide isreplaced with a neutral 2-amino ethylglycine moiety capable of forming apolyamide backbone which is highly resistant to nuclease digestion andimparts improved binding specificity to the molecule (Nielsen, et al.,Science, (2001), 254: 1497-1500).

Also preferred are nucleobase-modified ribonucleotides, i.e.,ribonucleotides, containing at least one non-naturally occurringnucleobase instead of a naturally occurring nucleobase. Bases may bemodified to block the activity of adenosine deaminase. Exemplarymodified nucleobases include, but are not limited to, uridine and/orcytidine modified at the 5-position, e.g., 5-(2-amino)propyl uridine,5-bromo uridine; adenosine and/or guanosines modified at the 8 position,e.g., 8-bromo guanosine; deazanucleotides, e.g., 7-deaza-adenosine; O-and N-alkylated nucleotides, e.g., N6-methyl adenosine are suitable. Itshould be noted that the above modifications may be combined.

In other embodiments, cross-linking can be employed to alter thepharmacokinetics of the RNA silencing agent, for example, to increasehalf-life in the body. Thus, the invention includes RNA silencing agentshaving two complementary strands of nucleic acid, wherein the twostrands are crosslinked. The invention also includes RNA silencingagents which are conjugated or unconjugated (e.g., at its 3′ terminus)to another moiety (e.g. a non-nucleic acid moiety such as a peptide), anorganic compound (e.g., a dye), or the like). Modifying siRNAderivatives in this way may improve cellular uptake or enhance cellulartargeting activities of the resulting siRNA derivative as compared tothe corresponding siRNA, are useful for tracing the siRNA derivative inthe cell, or improve the stability of the siRNA derivative compared tothe corresponding siRNA.

Other exemplary modifications include: (a) 2′ modification, e.g.,provision of a 2′ OMe moiety on a U in a sense or antisense strand, butespecially on a sense strand, or provision of a 2′ OMe moiety in a 3′overhang, e.g., at the 3′ terminus (3′ terminus means at the 3′ atom ofthe molecule or at the most 3′ moiety, e.g., the most 3′P or 2′position, as indicated by the context); (b) modification of thebackbone, e.g., with the replacement of an 0 with an S, in the phosphatebackbone, e.g., the provision of a phosphorothioate modification, on theU or the A or both, especially on an antisense strand; e.g., with thereplacement of a O with an S; (c) replacement of the U with a C5 aminolinker; (d) replacement of an A with a G (sequence changes are preferredto be located on the sense strand and not the antisense strand); and (d)modification at the 2′, 6′, 7′, or 8′ position. Exemplary embodimentsare those in which one or more of these modifications are present on thesense but not the antisense strand, or embodiments where the antisensestrand has fewer of such modifications. Yet other exemplarymodifications include the use of a methylated P in a 3′ overhang, e.g.,at the 3′ terminus; combination of a 2′ modification, e.g., provision ofa 2′O Me moiety and modification of the backbone, e.g., with thereplacement of a O with an S, e.g., the provision of a phosphorothioatemodification, or the use of a methylated P, in a 3′ overhang, e.g., atthe 3′ terminus; modification with a 3′ alkyl; modification with anabasic pyrrolidone in a 3′ overhang, e.g., at the 3′ terminus;modification with naproxen, ibuprofen, or other moieties which inhibitdegradation at the 3′ terminus.

4) Modifications to Enhance Cellular Uptake

In other embodiments, RNA silencing agents may be modified with chemicalmoieties, for example, to enhance cellular uptake by target cells (e.g.,neuronal cells). Thus, the invention includes RNA silencing agents whichare conjugated or unconjugated (e.g., at its 3′ terminus) to anothermoiety (e.g. a non-nucleic acid moiety such as a peptide), an organiccompound (e.g., a dye), or the like. The conjugation can be accomplishedby methods known in the art, e.g., using the methods of Lambert et al.,Drug Deliv. Rev.: 47(1), 99-112 (2001) (describes nucleic acids loadedto polyalkylcyanoacrylate (PACA) nanoparticles); Fattal et al., J.Control Release 53(1-3):137-43 (1998) (describes nucleic acids bound tonanoparticles); Schwab et al., Ann. Oncol. 5 Suppl. 4:55-8 (1994)(describes nucleic acids linked to intercalating agents, hydrophobicgroups, polycations or PACA nanoparticles); and Godard et al., Eur. J.Biochem. 232(2):404-10 (1995) (describes nucleic acids linked tonanoparticles).

In a particular embodiment, an RNA silencing agent of invention isconjugated to a lipophilic moiety. In one embodiment, the lipophilicmoiety is a ligand that includes a cationic group. In anotherembodiment, the lipophilic moiety is attached to one or both strands ofan siRNA. In an exemplary embodiment, the lipophilic moiety is attachedto one end of the sense strand of the siRNA. In another exemplaryembodiment, the lipophilic moiety is attached to the 3′ end of the sensestrand. In some embodiments, the lipophilic moiety is selected from thegroup consisting of cholesterol, vitamin E, vitamin K, vitamin A, folicacid, or a cationic dye (e.g., Cy3). In an exemplary embodiment, thelipophilic moiety is a cholesterol. Other lipophilic moieties includecholic acid, adamantane acetic acid, 1-pyrene butyric acid,dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexylgroup, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecylgroup, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid,O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.

5) Tethered Ligands

Other entities can be tethered to an RNA silencing agent of theinvention. For example, a ligand tethered to an RNA silencing agent toimprove stability, hybridization thermodynamics with a target nucleicacid, targeting to a particular tissue or cell-type, or cellpermeability, e.g., by an endocytosis-dependent or -independentmechanism. Ligands and associated modifications can also increasesequence specificity and consequently decrease off-site targeting. Atethered ligand can include one or more modified bases or sugars thatcan function as intercalators. These are preferably located in aninternal region, such as in a bulge of RNA silencing agent/targetduplex. The intercalator can be an aromatic, e.g., a polycyclic aromaticor heterocyclic aromatic compound. A polycyclic intercalator can havestacking capabilities, and can include systems with 2, 3, or 4 fusedrings. The universal bases described herein can be included on a ligand.In one embodiment, the ligand can include a cleaving group thatcontributes to target gene inhibition by cleavage of the target nucleicacid. The cleaving group can be, for example, a bleomycin (e.g.,bleomycin-A5, bleomycin-A2, or bleomycin-B2), pyrene, phenanthroline(e.g., O-phenanthroline), a polyamine, a tripeptide (e.g., lys-tyr-lystripeptide), or metal ion chelating group. The metal ion chelating groupcan include, e.g., an Lu(III) or EU(III) macrocyclic complex, a Zn(II)2,9-dimethylphenanthroline derivative, a Cu(II) terpyridine, oracridine, which can promote the selective cleavage of target RNA at thesite of the bulge by free metal ions, such as Lu(III). In someembodiments, a peptide ligand can be tethered to a RNA silencing agentto promote cleavage of the target RNA, e.g., at the bulge region. Forexample, 1,8-dimethyl-1,3,6,8,10,13-hexaazacyclotetradecane (cyclam) canbe conjugated to a peptide (e.g., by an amino acid derivative) topromote target RNA cleavage. A tethered ligand can be an aminoglycosideligand, which can cause an RNA silencing agent to have improvedhybridization properties or improved sequence specificity. Exemplaryaminoglycosides include glycosylated polylysine, galactosylatedpolylysine, neomycin B, tobramycin, kanamycin A, and acridine conjugatesof aminoglycosides, such as Neo-N-acridine, Neo-S-acridine,Neo-C-acridine, Tobra-N-acridine, and KanaA-N-acridine. Use of anacridine analog can increase sequence specificity. For example, neomycinB has a high affinity for RNA as compared to DNA, but lowsequence-specificity. An acridine analog, neo-5-acridine has anincreased affinity for the HIV Rev-response element (RRE). In someembodiments the guanidine analog (the guanidinoglycoside) of anaminoglycoside ligand is tethered to an RNA silencing agent. In aguanidinoglycoside, the amine group on the amino acid is exchanged for aguanidine group. Attachment of a guanidine analog can enhance cellpermeability of an RNA silencing agent. A tethered ligand can be apoly-arginine peptide, peptoid or peptidomimetic, which can enhance thecellular uptake of an oligonucleotide agent.

Exemplary ligands are coupled, preferably covalently, either directly orindirectly via an intervening tether, to a ligand-conjugated carrier. Inexemplary embodiments, the ligand is attached to the carrier via anintervening tether. In exemplary embodiments, a ligand alters thedistribution, targeting or lifetime of an RNA silencing agent into whichit is incorporated. In exemplary embodiments, a ligand provides anenhanced affinity for a selected target, e.g., molecule, cell or celltype, compartment, e.g., a cellular or organ compartment, tissue, organor region of the body, as, e.g., compared to a species absent such aligand.

Exemplary ligands can improve transport, hybridization, and specificityproperties and may also improve nuclease resistance of the resultantnatural or modified RNA silencing agent, or a polymeric moleculecomprising any combination of monomers described herein and/or naturalor modified ribonucleotides. Ligands in general can include therapeuticmodifiers, e.g., for enhancing uptake; diagnostic compounds or reportergroups e.g., for monitoring distribution; cross-linking agents;nuclease-resistance conferring moieties; and natural or unusualnucleobases. General examples include lipophiles, lipids, steroids(e.g., uvaol, hecigenin, diosgenin), terpenes (e.g., triterpenes, e.g.,sarsasapogenin, Friedelin, epifriedelanol derivatized lithocholic acid),vitamins (e.g., folic acid, vitamin A, biotin, pyridoxal),carbohydrates, proteins, protein binding agents, integrin targetingmolecules, polycationics, peptides, polyamines, and peptide mimics.Ligands can include a naturally occurring substance, (e.g., human serumalbumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate(e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin orhyaluronic acid); amino acid, or a lipid. The ligand may also be arecombinant or synthetic molecule, such as a synthetic polymer, e.g., asynthetic polyamino acid. Examples of polyamino acids include polyaminoacid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid,styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied)copolymer, divinyl ether-maleic anhydride copolymer,N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol(PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllicacid), N-isopropylacrylamide polymers, or polyphosphazine. Example ofpolyamines include: polyethylenimine, polylysine (PLL), spermine,spermidine, polyamine, pseudopeptide-polyamine, peptidomimeticpolyamine, dendrimer polyamine, arginine, amidine, protamine, cationiclipid, cationic porphyrin, quaternary salt of a polyamine, or an alphahelical peptide.

Ligands can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a kidney cell.A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, mucin carbohydrate, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-glucosamine, multivalent mannose, multivalent fucose,glycosylated polyaminoacids, multivalent galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, biotin, or an RGD peptide orRGD peptide mimetic. Other examples of ligands include dyes,intercalating agents (e.g. acridines and substituted acridines),cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4,texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g.,phenazine, dihydrophenazine, phenanthroline, pyrenes), lys-tyr-lystripeptide, aminoglycosides, guanidium aminoglycodies, artificialendonucleases (e.g. EDTA), lipophilic molecules, e.g, cholesterol (andthio analogs thereof), cholic acid, cholanic acid, lithocholic acid,adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone,glycerol (e.g., esters (e.g., mono, bis, or tris fatty acid esters,e.g., C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, or C₂₀ fattyacids) and ethers thereof, e.g., C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇,C₁₈, C₁₉, or C₂₀ alkyl; e.g., 1,3-bis-O(hexadecyl)glycerol,1,3-bis-O(octadecyl)glycerol), geranyloxyhexyl group, hexadecylglycerol,bomeol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,stearic acid (e.g., glyceryl distearate), oleic acid, myristic acid,O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl,or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tatpeptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g.,PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl,radiolabeled markers, enzymes, haptens (e.g. biotin),transport/absorption facilitators (e.g., aspirin, naproxen, vitamin E,folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole,histamine, imidazole clusters, acridine-imidazole conjugates, Eu³⁺complexes of tetraazamacrocycles), dinitrophenyl, HRP or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g.,molecules having a specific affinity for a co-ligand, or antibodiese.g., an antibody, that binds to a specified cell type such as a cancercell, endothelial cell, or bone cell. Ligands may also include hormonesand hormone receptors. They can also include non-peptidic species, suchas lipids, lectins, carbohydrates, vitamins, cofactors, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-glucosamine multivalent mannose, or multivalent fucose. Theligand can be, for example, a lipopolysaccharide, an activator of p38MAP kinase, or an activator of NF-κB.

The ligand can be a substance, e.g., a drug, which can increase theuptake of the RNA silencing agent into the cell, for example, bydisrupting the cell's cytoskeleton, e.g., by disrupting the cell'smicrotubules, microfilaments, and/or intermediate filaments. The drugcan be, for example, taxon, vincristine, vinblastine, cytochalasin,nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A,indanocine, or myoservin. The ligand can increase the uptake of the RNAsilencing agent into the cell by activating an inflammatory response,for example. Exemplary ligands that would have such an effect includetumor necrosis factor alpha (TNFα), interleukin-1 beta, or gammainterferon. In one aspect, the ligand is a lipid or lipid-basedmolecule. Such a lipid or lipid-based molecule preferably binds a serumprotein, e.g., human serum albumin (HSA). An HSA binding ligand allowsfor distribution of the conjugate to a target tissue, e.g., a non-kidneytarget tissue of the body. For example, the target tissue can be theliver, including parenchymal cells of the liver. Other molecules thatcan bind HSA can also be used as ligands. For example, neproxin oraspirin can be used. A lipid or lipid-based ligand can (a) increaseresistance to degradation of the conjugate, (b) increase targeting ortransport into a target cell or cell membrane, and/or (c) can be used toadjust binding to a serum protein, e.g., HSA. A lipid based ligand canbe used to modulate, e.g., control the binding of the conjugate to atarget tissue. For example, a lipid or lipid-based ligand that binds toHSA more strongly will be less likely to be targeted to the kidney andtherefore less likely to be cleared from the body. A lipid orlipid-based ligand that binds to HSA less strongly can be used to targetthe conjugate to the kidney. In a preferred embodiment, the lipid basedligand binds HSA. A lipid-based ligand can bind HSA with a sufficientaffinity such that the conjugate will be preferably distributed toanon-kidney tissue. However, it is preferred that the affinity not be sostrong that the HSA-ligand binding cannot be reversed. In anotherpreferred embodiment, the lipid based ligand binds HSA weakly or not atall, such that the conjugate will be preferably distributed to thekidney. Other moieties that target to kidney cells can also be used inplace of or in addition to the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which istaken up by a target cell, e.g., a proliferating cell. These areparticularly useful for treating disorders characterized by unwantedcell proliferation, e.g., of the malignant or non-malignant type, e.g.,cancer cells. Exemplary vitamins include vitamin A, E, and K. Otherexemplary vitamins include are B vitamin, e.g., folic acid, B12,riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up bycancer cells. Also included are HSA and low density lipoprotein (LDL).

In another aspect, the ligand is a cell-permeation agent, preferably ahelical cell-permeation agent. Preferably, the agent is amphipathic. Anexemplary agent is a peptide such as tat or antennopedia. If the agentis a peptide, it can be modified, including a peptidylmimetic,invertomers, non-peptide or pseudo-peptide linkages, and use of D-aminoacids. The helical agent is preferably an alpha-helical agent, whichpreferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (alsoreferred to herein as an oligopeptidomimetic) is a molecule capable offolding into a defined three-dimensional structure similar to a naturalpeptide. The attachment of peptide and peptidomimetics tooligonucleotide agents can affect pharmacokinetic distribution of theRNA silencing agent, such as by enhancing cellular recognition andabsorption. The peptide or peptidomimetic moiety can be about 5-50 aminoacids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 aminoacids long. A peptide or peptidomimetic can be, for example, a cellpermeation peptide, cationic peptide, amphipathic peptide, orhydrophobic peptide (e.g., consisting primarily of Tyr, Trp or Phe). Thepeptide moiety can be a dendrimer peptide, constrained peptide orcrosslinked peptide. The peptide moiety can be an L-peptide orD-peptide. In another alternative, the peptide moiety can include ahydrophobic membrane translocation sequence (MTS). A peptide orpeptidomimetic can be encoded by a random sequence of DNA, such as apeptide identified from a phage-display library, orone-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature354:82-84, 1991). In exemplary embodiments, the peptide orpeptidomimetic tethered to an RNA silencing agent via an incorporatedmonomer unit is a cell targeting peptide such as anarginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. A peptidemoiety can range in length from about 5 amino acids to about 40 aminoacids. The peptide moieties can have a structural modification, such asto increase stability or direct conformational properties. Any of thestructural modifications described below can be utilized.

6) Branched Oligonucleotides

Two or more oligonucleotides, where at least one of the oligonucleotidesincludes an intersubunit linkage according to an embodiment of Formula(I), may be connected to one another by one or more moietiesindependently selected from a linker, a spacer and a branching point, toform a branched compound. For example, the branched compound may containtwo or more RNA silencing agents of the types set out above, resultingin a new type of RNA silencing agent having a branched structure. Inrepresentative embodiments, the oligonucleotides each comprise anantisense strand (or portions thereof), wherein the antisense strand hassufficient complementary to a heterozygous single nucleotidepolymorphism to mediate an RNA-mediated silencing mechanism (e.g. RNAi).

In exemplary embodiments, the branched compounds may have two to eightRNA silencing agents attached through a linker. The linker may behydrophobic. In a typical embodiment, branched oligonucleotides of thepresent application have two to three oligonucleotides. In someembodiments, the oligonucleotides independently have substantialchemical stabilization (e.g., at least 40% of the constituent bases arechemically-modified). In a particular embodiment, the oligonucleotideshave full chemical stabilization (i.e., all the constituent bases arechemically-modified). In some embodiments, branched oligonucleotidescomprise one or more single-stranded phosphorothioated tails, each tailindependently having two to twenty nucleotides. In a non-limitingembodiment, each single-stranded tail has eight to ten nucleotides.

In some embodiments, branched compounds are characterized by threeproperties: (1) a branched structure, (2) full metabolic stabilization,and (3) the presence of a single-stranded tail comprisingphosphorothioate linkers. In an exemplary embodiment, branchedoligonucleotides have 2 or 3 branches. The increased overall size of thebranched structures promotes increased uptake. Also, without being boundby a particular theory of activity, it appears that multiple adjacentbranches (e.g., 2 or 3) allow each branch to act cooperatively and thusdramatically enhance rates of internalization, trafficking and release.

Branched compounds are provided in various structurally diverseembodiments. As shown in FIG. 24, for example, in some embodimentsnucleic acids attached at the branching points are single stranded andinclude miRNA inhibitors, gapmers, mixmers, SSOs, PMOs, or PNAs. Thesesingle strands can be attached at their 3′ or 5′ end. Combinations ofsiRNA and single stranded oligonucleotides could also be used for dualfunction. In another embodiment, short nucleic acids complementary tothe gapmers, mixmers, miRNA inhibitors, SSOs, PMOs, and PNAs are used tocarry these active single-stranded nucleic acids and enhancedistribution and cellular internalization. The short duplex region has alow melting temperature (Tm 37° C.) for fast dissociation uponinternalization of the branched structure into the cell.

As shown in FIG. 33, “Di-siRNA” compounds, that is, branchedoligonucleotides having two siRNAs and a linker, may comprise chemicallydiverse conjugates. Conjugated bioactive ligands may be used to enhancecellular specificity and to promote membrane association,internalization, and serum protein binding. Examples of bioactivemoieties to be used for conjugation include DHAg2, DHA, GalNAc, andcholesterol. These moieties can be attached to Di-siRNA either throughthe connecting linker or spacer, or added via an additional linker orspacer attached to another free siRNA end.

Without being bound to any particular theory, it has been found that thepresence of a branched structure improves the level of tissue retentionin the brain more than 100-fold compared to non-branched compounds ofidentical chemical composition, suggesting a new mechanism of cellularretention and distribution. Branched oligonucleotides have unexpectedlyuniform distribution throughout the spinal cord and brain. Moreover,branched oligonucleotides exhibit unexpectedly efficient systemicdelivery to a variety of tissues, and very high levels of tissueaccumulation.

Branched oligonucleotides may comprise a variety of therapeutic nucleicacids, including ASOs, miRNAs, miRNA inhibitors, splice switching, PMOs,PNAs. In some embodiments, branched oligonucleotides further compriseconjugated hydrophobic moieties and exhibit unprecedented silencing andefficacy in vitro and in vivo.

Non-limiting embodiments of branched oligonucleotide configurations aredisclosed in FIGS. 18, 24-26, 32-34, and 57-62. Non-limiting examples oflinkers, spacers and branching points are disclosed in FIG. 24.

Linkers

In some embodiments of the branched oligonucleotide compounds, eachlinker is independently selected from an ethylene glycol chain, an alkylchain, a peptide, RNA, DNA, a phosphate, a phosphonate, aphosphoramidate, an ester, an amide, a triazole, and combinationsthereof; wherein any carbon or oxygen atom of the linker is optionallyreplaced with a nitrogen atom, bears a hydroxyl substituent, or bears anoxo substituent. In one embodiment, each linker is an ethylene glycolchain. In another embodiment, each linker is an alkyl chain. In anotherembodiment, each linker is a peptide. In another embodiment, each linkeris RNA. In another embodiment, each linker is DNA. In anotherembodiment, each linker is a phosphate. In another embodiment, eachlinker is a phosphonate. In another embodiment, each linker is aphosphoramidate. In another embodiment, each linker is an ester. Inanother embodiment, each linker is an amide. In another embodiment, eachlinker is a triazole. In another embodiment, each linker is a structureselected from the formulas of FIG. 23.

VI. Compound of Formula (1)

In another aspect, provided herein is a branched oligonucleotidecompound of formula (1):

L-(N)_(n)   (1)

wherein L is selected from an ethylene glycol chain, an alkyl chain, apeptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, anester, an amide, a triazole, and combinations thereof, wherein Formula(1) optionally further comprises one or more branch point Bp, and one ormore spacer S; wherein Bp is independently for each occurrence apolyvalent organic species or derivative thereof; S is independently foreach occurrence selected from an ethylene glycol chain, an alkyl chain,a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, anester, an amide, a triazole, and combinations thereof; N is an RNAduplex comprising a sense strand and an antisense strand, wherein thesense strand and antisense strand each independently comprise one ormore chemical modifications; and n is 2, 3, 4, 5, 6, 7 or 8. In someembodiments, at least one N includes a modified intersubunit linkage ofFormula (I).

In some embodiments, the compound of Formula (1) has a structureselected from formulas (1-1)-(1-9) of Table 1.

TABLE 1 N—L—N (1-1) N—S—L—S—N (1-2)

In one embodiment, the compound of Formula (1) is formula (1-1). Inanother embodiment, the compound of Formula (1) is Formula (1-2). Inanother embodiment, the compound of Formula (1) is Formula (1-3). Inanother embodiment, the compound of formula (1) is Formula (1-4). Inanother embodiment, the compound of Formula (1) is formula (1-5). Inanother embodiment, the compound of Formula (1) is Formula (1-6). Inanother embodiment, the compound of Formula (1) is Formula (1-7). Inanother embodiment, the compound of Formula (1) is Formula (1-8). Inanother embodiment, the compound of formula (1) is formula (1-9).

In some embodiments of the compound of Formula (1), each linker isindependently selected from an ethylene glycol chain, an alkyl chain, apeptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, anester, an amide, a triazole, and combinations thereof; wherein anycarbon or oxygen atom of the linker is optionally replaced with anitrogen atom, bears a hydroxyl substituent, or bears an oxosubstituent. In one embodiment of the compound of Formula (1), eachlinker is an ethylene glycol chain. In another embodiment, each linkeris an alkyl chain. In another embodiment of the compound of Formula (1),each linker is a peptide. In another embodiment of the compound ofFormula (1), each linker is RNA. In another embodiment of the compoundof Formula (1), each linker is DNA. In another embodiment of thecompound of Formula (1), each linker is a phosphate. In anotherembodiment, each linker is a phosphonate. In another embodiment of thecompound of Formula (1), each linker is a phosphoramidate. In anotherembodiment of the compound of Formula (1), each linker is an ester. Inanother embodiment of the compound of Formula (1), each linker is anamide. In another embodiment of the compound of Formula (1), each linkeris a triazole. In another embodiment of the compound of Formula (1),each linker is a structure selected from the formulas of FIG. 23.

In one embodiment of the compound of Formula (1), Bp is a polyvalentorganic species. In another embodiment of the compound of Formula (1),Bp is a derivative of a polyvalent organic species. In one embodiment ofthe compound of Formula (1), Bp is a triol or tetrol derivative. Inanother embodiment, Bp is a tri- or tetra-carboxylic acid derivative. Inanother embodiment, Bp is an amine derivative. In another embodiment, Bpis a tri- or tetra-amine derivative. In another embodiment, Bp is anamino acid derivative. In another embodiment of the compound of Formula(1), Bp is selected from the formulas of FIG. 23.

Polyvalent organic species are moieties comprising carbon and three ormore valencies (i.e., points of attachment with moieties such as S, L orN, as defined above). Non-limiting examples of polyvalent organicspecies include triols (e.g., glycerol, phloroglucinol, and the like),tetrols (e.g., ribose, pentaerythritol, 1,2,3,5-tetrahydroxybenzene, andthe like), tri-carboxylic acids (e.g., citric acid,1,3,5-cyclohexanetricarboxylic acid, trimesic acid, and the like),tetra-carboxylic acids (e.g., ethylenediaminetetraacetic acid,pyromellitic acid, and the like), tertiary amines (e.g.,tripropargylamine, triethanolamine, and the like), triamines (e.g.,diethylenetriamine and the like), tetramines, and species comprising acombination of hydroxyl, thiol, amino, and/or carboxyl moieties (e.g.,amino acids such as lysine, serine, cysteine, and the like).

In some embodiments of the compound of Formula (1), each nucleic acidcomprises one or more chemically-modified nucleotides. In someembodiments of the compound of Formula (1), each nucleic acid consistsof chemically-modified nucleotides. In some embodiments of the compoundof Formula (1), >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55%or >50% of each nucleic acid comprises chemically-modified nucleotides.

In some embodiments, each antisense strand independently comprises a 5′terminal group R selected from the groups of Table 2.

TABLE 2

In one embodiment, R is R₁. In another embodiment, R is R₂. In anotherembodiment, R is R₃. In another embodiment, R is R₄. In anotherembodiment, R is R₅. In another embodiment, R is R₆. In anotherembodiment, R is R₇. In another embodiment, R is R₈.

Structure of Formula (2)

In some embodiments, the compound of Formula (1) the structure ofFormula (2):

wherein X, for each occurrence, independently, is selected fromadenosine, guanosine, uridine, cytidine, and chemically-modifiedderivatives thereof; Y, for each occurrence, independently, is selectedfrom adenosine, guanosine, uridine, cytidine, and chemically-modifiedderivatives thereof; - represents a phosphodiester internucleosidelinkage; = represents a phosphorothioate internucleoside linkage; and--- represents, individually for each occurrence, a base-pairinginteraction or a mismatch. Moreover, at least one of the internucleosidelinkages may be replaced with a modified intersubunit linkage of Formula(I).

In some embodiments, the structure of Formula (2) does not containmismatches. In one embodiment, the structure of Formula (2) contains 1mismatch. In another embodiment, the compound of Formula (2) contains 2mismatches. In another embodiment, the compound of Formula (2) contains3 mismatches. In another embodiment, the compound of Formula (2)contains 4 mismatches. In some embodiments, each nucleic acid consistsof chemically-modified nucleotides.

In someembodiments, >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55%or >50% of X's of the structure of Formula (2) are chemically-modifiednucleotides. In otherembodiments, >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55%or >50% of X's of the structure of Formula (2) are chemically-modifiednucleotides.

Structure of Formula (3)

In some embodiments, the compound of Formula (1) has the structure ofFormula (3):

wherein X, for each occurrence, independently, is a nucleotidecomprising a 2′-deoxy-2′-fluoro modification; X, for each occurrence,independently, is a nucleotide comprising a 2′-O-methyl modification; Yfor each occurrence, independently, is a nucleotide comprising a2′-deoxy-2′-fluoro modification; and Y, for each occurrence,independently, is a nucleotide comprising a 2′-O-methyl modification.

In some embodiments, X is chosen from the group consisting of2′-deoxy-2′-fluoro modified adenosine, guanosine, uridine or cytidine.In some embodiments, X is chosen from the group consisting of2′-O-methyl modified adenosine, guanosine, uridine or cytidine. In someembodiments, Y is chosen from the group consisting of 2′-deoxy-2′-fluoromodified adenosine, guanosine, uridine or cytidine. In some embodiments,Y is chosen from the group consisting of 2′-O-methyl modified adenosine,guanosine, uridine or cytidine.

In some embodiments, the structure of Formula (3) does not containmismatches. In one embodiment, the structure of Formula (3) contains 1mismatch. In another embodiment, the compound of Formula (3) contains 2mismatches. In another embodiment, the compound of Formula (3) contains3 mismatches. In another embodiment, the compound of Formula (3)contains 4 mismatches.

Structure of Formula (4)

In some embodiments, the compound of Formula (1) has the structure ofFormula (4):

wherein X, for each occurrence, independently, is selected fromadenosine, guanosine, uridine, cytidine, and chemically-modifiedderivatives thereof; Y, for each occurrence, independently, is selectedfrom adenosine, guanosine, uridine, cytidine, and chemically-modifiedderivatives thereof; - represents a phosphodiester internucleosidelinkage; = represents a phosphorothioate internucleoside linkage; and--- represents, individually for each occurrence, a base-pairinginteraction or a mismatch. Also, at least one of the internucleosidelinkages may be replaced with a modified intersubunit linkage of Formula(I).

In some embodiments, the structure of Formula (4) does not containmismatches. In one embodiment, the structure of Formula (4) contains 1mismatch. In another embodiment, the compound of Formula (4) contains 2mismatches. In another embodiment, the compound of Formula (4) contains3 mismatches. In another embodiment, the compound of Formula (4)contains 4 mismatches. In some embodiments, each nucleic acid consistsof chemically-modified nucleotides.

In someembodiments, >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55%or >50% of X's of the structure of Formula (2) are chemically-modifiednucleotides. In otherembodiments, >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55%or >50% of X's of the structure of Formula (2) are chemically-modifiednucleotides.

Structure of Formula (5)

In some embodiments, the compound of Formula (1) has the structure ofFormula (5):

wherein X, for each occurrence, independently, is a nucleotidecomprising a 2′-deoxy-2′-fluoro modification; X, for each occurrence,independently, is a nucleotide comprising a 2′-O-methyl modification; Yfor each occurrence, independently, is a nucleotide comprising a2′-deoxy-2′-fluoro modification; and Y, for each occurrence,independently, is a nucleotide comprising a 2′-O-methyl modification.

In some embodiments, X is chosen from the group consisting of2′-deoxy-2′-fluoro modified adenosine, guanosine, uridine or cytidine.In some embodiments, X is chosen from the group consisting of2′-O-methyl modified adenosine, guanosine, uridine or cytidine. In someembodiments, Y is chosen from the group consisting of 2′-deoxy-2′-fluoromodified adenosine, guanosine, uridine or cytidine. In some embodiments,Y is chosen from the group consisting of 2′-O-methyl modified adenosine,guanosine, uridine or cytidine.

In some embodiments, the structure of Formula (5) does not containmismatches. In one embodiment, the structure of Formula (6) contains 1mismatch. In another embodiment, the compound of Formula (5) contains 2mismatches. In another embodiment, the compound of Formula (5) contains3 mismatches. In another embodiment, the compound of formula (V)contains 4 mismatches.

Variable Linkers

In some embodiments of the compound of Formula (1), L has the structureof L1:

In some embodiments of L1, R is R³ and n is 2.

In some embodiments of the structure of formula (II), L has thestructure of L1. In some embodiments of the structure of formula (III),L has the structure of L1. In some embodiments of the structure offormula (IV), L has the structure of L1. In some embodiments of thestructure of formula (V), L has the structure of L1. In some embodimentsof the structure of formula (VI), L has the structure of L1. In someembodiments of the structure of formula (VII), L has the structure ofL1.

In some embodiments of the compound of Formula (1), L has the structureof L2:

In some embodiments of L2, R is R³ and n is 2. In some embodiments ofthe structure of Formula (2), L has the structure of L2. In someembodiments of the structure of Formula (3), L has the structure of L2.In some embodiments of the structure of Formula (4), L has the structureof L2. In some embodiments of the structure of Formula (5), L has thestructure of L2.

Delivery System

In another aspect, provided herein is a delivery system for therapeuticnucleic acids having the structure of Formula (6):

L-(cNA)_(n)   (6)

wherein L is selected from an ethylene glycol chain, an alkyl chain, apeptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, anester, an amide, a triazole, and combinations thereof, wherein Formula(6) optionally further comprises one or more branch point Bp, and one ormore spacer S; wherein Bp is independently for each occurrence apolyvalent organic species or derivative thereof; S is independently foreach occurrence selected from an ethylene glycol chain, an alkyl chain,a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, anester, an amide, a triazole, and combinations thereof; each cNA,independently, is a carrier nucleic acid comprising one or more chemicalmodifications; and n is 2, 3, 4, 5, 6, 7 or 8. In some embodiments, atleast one cNA includes a modified intersubunit linkage of Formula (I).

In one embodiment of the delivery system, L is an ethylene glycol chain.In another embodiment of the delivery system, L is an alkyl chain. Inanother embodiment of the delivery system, L is a peptide. In anotherembodiment of the delivery system, L is RNA. In another embodiment ofthe delivery system, L is DNA. In another embodiment of the deliverysystem, L is a phosphate. In another embodiment of the delivery system,L is a phosphonate. In another embodiment of the delivery system, L is aphosphoramidate. In another embodiment of the delivery system, L is anester. In another embodiment of the delivery system, L is an amide. Inanother embodiment of the delivery system, L is a triazole.

In one embodiment of the delivery system, S is an ethylene glycol chain.In another embodiment, S is an alkyl chain. In another embodiment of thedelivery system, S is a peptide. In another embodiment, S is RNA. Inanother embodiment of the delivery system, S is DNA. In anotherembodiment of the delivery system, S is a phosphate. In anotherembodiment of the delivery system, S is a phosphonate. In anotherembodiment of the delivery system, S is a phosphoramidate. In anotherembodiment of the delivery system, S is an ester. In another embodiment,S is an amide. In another embodiment, S is a triazole.

In one embodiment of the delivery system, n is 2. In another embodimentof the delivery system, n is 3. In another embodiment of the deliverysystem, n is 4. In another embodiment of the delivery system, n is 5. Inanother embodiment of the delivery system, n is 6. In another embodimentof the delivery system, n is 7. In another embodiment of the deliverysystem, n is 8.

In some embodiments, each cNAcomprises >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55% or >50%chemically-modified nucleotides.

In some embodiments, the compound of Formula (6) has a structureselected from formulas (6-1)-(6-9) of Table 3:

TABLE 3 ANc—L—cNA (6-1) ANc—S—L—S—cNA (6-2)

In some embodiments, the compound of Formula (6) is the structure offormula (6-1). In some embodiments, the compound of Formula (6) is thestructure of Formula (6-2). In some embodiments, the compound of Formula(6) is the structure of Formula (6-3). In some embodiments, the compoundof Formula (6) is the structure of Formula (6-4). In some embodiments,the compound of Formula (6) is the structure of formula (6-5). In someembodiments, the compound of Formula (6) is the structure of formula(6-6). In some embodiments, the compound of Formula (6) is the structureof Formula (6-7). In some embodiments, the compound of Formula (6) isthe structure of formula (6-8). In some embodiments, the compound ofFormula (6) is the structure of formula (6-9).

In some embodiments, the compound of Formulas (6) (including, e.g., oneof formulas (6-1)-(6-9), each cNA independently comprises at least 15contiguous nucleotides. In some embodiments, each cNA independentlyconsists of chemically-modified nucleotides.

In some embodiments, each NA is hybridized to at least one cNA. In someembodiments, at least one NA includes a modified intersubunit linkage ofFormula (I). In some embodiments, compounds of the invention arecharacterized by the following properties: (1) two or more branchedoligonucleotides, e.g., wherein there is a non-equal number of 3′ and 5′ends; (2) substantially chemically stabilized, e.g., wherein more than40%, optimally 100%, of oligonucleotides are chemically modified (e.g.,no RNA and optionally no DNA); and (3) phosphorothioated singleoligonucleotides containing at least 3, optimally 5-20 phosphorothioatedbonds.

VII. Methods of Introducing Nucleic Acids, Vectors Host Cells, andBranched Oligonucleotide Compounds

RNA silencing agents of the invention may be directly introduced intothe cell (e.g., a neural cell) (i.e., intracellularly); or introducedextracellularly into a cavity, interstitial space, into the circulationof an organism, introduced orally, or may be introduced by bathing acell or organism in a solution containing the nucleic acid. Vascular orextravascular circulation, the blood or lymph system, and thecerebrospinal fluid are sites where the nucleic acid may be introduced.

The RNA silencing agents of the invention can be introduced usingnucleic acid delivery methods known in art including injection of asolution containing the nucleic acid, bombardment by particles coveredby the nucleic acid, soaking the cell or organism in a solution of thenucleic acid, or electroporation of cell membranes in the presence ofthe nucleic acid. Other methods known in the art for introducing nucleicacids to cells may be used, such as lipid-mediated carrier transport,chemical-mediated transport, and cationic liposome transfection such ascalcium phosphate, and the like. The nucleic acid may be introducedalong with other components that perform one or more of the followingactivities: enhance nucleic acid uptake by the cell or other-wiseincrease inhibition of the target gene.

Physical methods of introducing nucleic acids include injection of asolution containing the RNA, bombardment by particles covered by theRNA, soaking the cell or organism in a solution of the RNA, orelectroporation of cell membranes in the presence of the RNA. A viralconstruct packaged into a viral particle would accomplish both efficientintroduction of an expression construct into the cell and transcriptionof RNA encoded by the expression construct. Other methods known in theart for introducing nucleic acids to cells may be used, such aslipid-mediated carrier transport, chemical-mediated transport, such ascalcium phosphate, and the like. Thus the RNA may be introduced alongwith components that perform one or more of the following activities:enhance RNA uptake by the cell, inhibit annealing of single strands,stabilize the single strands, or other-wise increase inhibition of thetarget gene.

RNA may be directly introduced into the cell (i.e., intracellularly); orintroduced extracellularly into a cavity, interstitial space, into thecirculation of an organism, introduced orally, or may be introduced bybathing a cell or organism in a solution containing the RNA. Vascular orextravascular circulation, the blood or lymph system, and thecerebrospinal fluid are sites where the RNA may be introduced.

The cell having the target gene may be from the germ line or somatic,totipotent or pluripotent, dividing or non-dividing, parenchyma orepithelium, immortalized or transformed, or the like. The cell may be astem cell or a differentiated cell. Cell types that are differentiatedinclude adipocytes, fibroblasts, myocytes, cardiomyocytes, endothelium,neurons, glia, blood cells, megakaryocytes, lymphocytes, macrophages,neutrophils, eosinophils, basophils, mast cells, leukocytes,granulocytes, keratinocytes, chondrocytes, osteoblasts, osteoclasts,hepatocytes, and cells of the endocrine or exocrine glands.

Depending on the particular target gene and the dose of double strandedRNA material delivered, this process may provide partial or completeloss of function for the target gene. A reduction or loss of geneexpression in at least 50%, 60%, 70%, 80%, 90%, 95% or 99% or more oftargeted cells is exemplary. Inhibition of gene expression refers to theabsence (or observable decrease) in the level of protein and/or mRNAproduct from a target gene. Specificity refers to the ability to inhibitthe target gene without manifest effects on other genes of the cell. Theconsequences of inhibition can be confirmed by examination of theoutward properties of the cell or organism (as presented below in theexamples) or by biochemical techniques such as RNA solutionhybridization, nuclease protection, Northern hybridization, reversetranscription, gene expression monitoring with a microarray, antibodybinding, Enzyme Linked ImmunoSorbent Assay (ELISA), Western blotting,RadioImmunoAssay (RIA), other immunoassays, and Fluorescence ActivatedCell Sorting (FACS).

For RNA-mediated inhibition in a cell line or whole organism, geneexpression is conveniently assayed by use of a reporter or drugresistance gene whose protein product is easily assayed. Such reportergenes include acetohydroxyacid synthase (AHAS), alkaline phosphatase(AP), beta galactosidase (LacZ), beta glucoronidase (GUS),chloramphenicol acetyltransferase (CAT), green fluorescent protein(GFP), horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase(NOS), octopine synthase (OCS), and derivatives thereof. Multipleselectable markers are available that confer resistance to ampicillin,bleomycin, chloramphenicol, gentarnycin, hygromycin, kanamycin,lincomycin, methotrexate, phosphinothricin, puromycin, and tetracyclin.Depending on the assay, quantitation of the amount of gene expressionallows one to determine a degree of inhibition which is greater than10%, 33%, 50%, 90%, 95% or 99% as compared to a cell not treatedaccording to the present invention. Lower doses of injected material andlonger times after administration of RNAi agent may result in inhibitionin a smaller fraction of cells (e.g., at least 10%, 20%, 50%, 75%, 90%,or 95% of targeted cells). Quantization of gene expression in a cell mayshow similar amounts of inhibition at the level of accumulation oftarget mRNA or translation of target protein. As an example, theefficiency of inhibition may be determined by assessing the amount ofgene product in the cell; mRNA may be detected with a hybridizationprobe having a nucleotide sequence outside the region used for theinhibitory double-stranded RNA, or translated polypeptide may bedetected with an antibody raised against the polypeptide sequence ofthat region.

The RNA may be introduced in an amount which allows delivery of at leastone copy per cell. Higher doses (e.g., at least 5, 10, 100, 500 or 1000copies per cell) of material may yield more effective inhibition; lowerdoses may also be useful for specific applications.

In an exemplary aspect, the efficacy of an RNAi agent of the invention(e.g., an siRNA targeting a target sequence of interest) is tested forits ability to specifically degrade mutant mRNA (e.g., target mRNAand/or the production of target protein) in cells, in particular, inneurons (e.g., striatal or cortical neuronal clonal lines and/or primaryneurons). Also suitable for cell-based validation assays are otherreadily transfectable cells, for example, HeLa cells or COS cells. Cellsare transfected with human wild type or mutant cDNAs (e.g., human wildtype or mutant target cDNA). Standard siRNA, modified siRNA or vectorsable to produce siRNA from U-looped mRNA are co-transfected. Selectivereduction in target mRNA and/or target protein is measured. Reduction oftarget mRNA or protein can be compared to levels of target mRNA orprotein in the absence of an RNAi agent or in the presence of an RNAiagent that does not target the target mRNA. Exogenously-introduced mRNAor protein (or endogenous mRNA or protein) can be assayed for comparisonpurposes. When utilizing neuronal cells, which are known to be somewhatresistant to standard transfection techniques, it may be desirable tointroduce RNAi agents (e.g., siRNAs) by passive uptake.

Recombinant Adeno-Associated Viruses and Vectors

In certain exemplary embodiments, recombinant adeno-associated viruses(rAAVs) and their associated vectors can be used to deliver one or moresiRNAs into cells, e.g., neural cells (e.g., brain cells). AAV is ableto infect many different cell types, although the infection efficiencyvaries based upon serotype, which is determined by the sequence of thecapsid protein. Several native AAV serotypes have been identified, withserotypes 1-9 being the most commonly used for recombinant AAV. AAV-2 isthe most well-studied and published serotype. The AAV-DJ system includesserotypes AAV-DJ and AAV-DJ/8. These serotypes were created through DNAshuffling of multiple AAV serotypes to produce AAV with hybrid capsidsthat have improved transduction efficiencies in vitro (AAV-DJ) and invivo (AAV-DJ/8) in a variety of cells and tissues.

In particular embodiments, widespread central nervous system (CNS)delivery can be achieved by intravascular delivery of recombinantadeno-associated virus 7 (rAAV7), RAAV9 and rAAV10, or other suitablerAAVs (Zhang et al. (2011) Mol. Ther. 19(8):1440-8. doi:10.1038/mt.2011.98. Epub 2011 May 24). rAAVs and their associatedvectors are well-known in the art and are described in US PatentApplications 2014/0296486, 2010/0186103, 2008/0269149, 2006/0078542 and2005/0220766, each of which is incorporated herein by reference in itsentirety for all purposes.

rAAVs may be delivered to a subject in compositions according to anyappropriate methods known in the art. An rAAV can be suspended in aphysiologically compatible carrier (i.e., in a composition), and may beadministered to a subject, i.e., a host animal, such as a human, mouse,rat, cat, dog, sheep, rabbit, horse, cow, goat, pig, guinea pig,hamster, chicken, turkey, a non-human primate (e.g., Macaque) or thelike. In some embodiments, a host animal is a non-human host animal.

Delivery of one or more rAAVs to a mammalian subject may be performed,for example, by intramuscular injection or by administration into thebloodstream of the mammalian subject. Administration into thebloodstream may be by injection into a vein, an artery, or any othervascular conduit. In some embodiments, one or more rAAVs areadministered into the bloodstream by way of isolated limb perfusion, atechnique well known in the surgical arts, the method essentiallyenabling the artisan to isolate a limb from the systemic circulationprior to administration of the rAAV virions. A variant of the isolatedlimb perfusion technique, described in U.S. Pat. No. 6,177,403, can alsobe employed by the skilled artisan to administer virions into thevasculature of an isolated limb to potentially enhance transduction intomuscle cells or tissue. Moreover, in certain instances, it may bedesirable to deliver virions to the central nervous system (CNS) of asubject. By “CNS” is meant all cells and tissue of the brain and spinalcord of a vertebrate. Thus, the term includes, but is not limited to,neuronal cells, glial cells, astrocytes, cerebrospinal fluid (CSF),interstitial spaces, bone, cartilage and the like. Recombinant AAVs maybe delivered directly to the CNS or brain by injection into, e.g., theventricular region, as well as to the striatum (e.g., the caudatenucleus or putamen of the striatum), spinal cord and neuromuscularjunction, or cerebellar lobule, with a needle, catheter or relateddevice, using neurosurgical techniques known in the art, such as bystereotactic injection (see, e.g., Stein et al., J Virol 73:3424-3429,1999; Davidson et al., PNAS 97:3428-3432, 2000; Davidson et al., Nat.Genet. 3:219-223, 1993; and Alisky and Davidson, Hum. Gene Ther.11:2315-2329, 2000).

The compositions of the invention may comprise an rAAV alone, or incombination with one or more other viruses (e.g., a second rAAV encodinghaving one or more different transgenes). In some embodiments, acomposition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or more different rAAVseach having one or more different transgenes.

An effective amount of an rAAV is an amount sufficient to target infectan animal, target a desired tissue. In some embodiments, an effectiveamount of an rAAV is an amount sufficient to produce a stable somatictransgenic animal model. The effective amount will depend primarily onfactors such as the species, age, weight, health of the subject, and thetissue to be targeted, and may thus vary among animal and tissue. Forexample, an effective amount of one or more rAAVs is generally in therange of from about 1 ml to about 100 ml of solution containing fromabout 109 to 10¹⁶ genome copies. In some cases, a dosage between about10¹¹ to 10¹² rAAV genome copies is appropriate. In some embodiments,10¹² rAAV genome copies is effective to target heart, liver, andpancreas tissues. In some cases, stable transgenic animals are producedby multiple doses of an rAAV.

In some embodiments, rAAV compositions are formulated to reduceaggregation of AAV particles in the composition, particularly where highrAAV concentrations are present (e.g., about 10¹³ genome copies/mL ormore). Methods for reducing aggregation of rAAVs are well known in theart and, include, for example, addition of surfactants, pH adjustment,salt concentration adjustment, etc. (See, e.g., Wright et al. (2005)Molecular Therapy 12:171-178, the contents of which are incorporatedherein by reference.)

“Recombinant AAV (rAAV) vectors” comprise, at a minimum, a transgene andits regulatory sequences, and 5′ and 3′ AAV inverted terminal repeats(ITRs). It is this recombinant AAV vector which is packaged into acapsid protein and delivered to a selected target cell. In someembodiments, the transgene is a nucleic acid sequence, heterologous tothe vector sequences, which encodes a polypeptide, protein, functionalRNA molecule (e.g., siRNA) or other gene product, of interest. Thenucleic acid coding sequence is operatively linked to regulatorycomponents in a manner which permits transgene transcription,translation, and/or expression in a cell of a target tissue.

The AAV sequences of the vector typically comprise the cis-acting 5′ and3′ inverted terminal repeat (ITR) sequences (See, e.g., B. J. Carter, in“Handbook of Parvoviruses”, ed., P. Tijsser, CRC Press, pp. 155 168(1990)). The ITR sequences are usually about 145 basepairs in length. Insome embodiments, substantially the entire sequences encoding the ITRsare used in the molecule, although some degree of minor modification ofthese sequences is permissible. The ability to modify these ITRsequences is within the skill of the art. (See, e.g., texts such asSambrook et al, “Molecular Cloning. A Laboratory Manual”, 2d ed., ColdSpring Harbor Laboratory, New York (1989); and K. Fisher et al., JVirol., 70:520 532 (1996)). An example of such a molecule employed inthe present invention is a “cis-acting” plasmid containing thetransgene, in which the selected transgene sequence and associatedregulatory elements are flanked by the 5′ and 3′ AAV ITR sequences. TheAAV ITR sequences may be obtained from any known AAV, includingmammalian AAV types described further herein.

VIII. Methods of Treatment

“Treatment,” or “treating,” as used herein, is defined as theapplication or administration of a therapeutic agent (e.g., a RNA agentor vector or transgene encoding same) to a patient, or application oradministration of a therapeutic agent to an isolated tissue or cell linefrom a patient, who has the disease or disorder, a symptom of disease ordisorder or a predisposition toward a disease or disorder, with thepurpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate,improve or affect the disease or disorder, the symptoms of the diseaseor disorder, or the predisposition toward disease.

In one aspect, the invention provides a method for preventing in asubject, a disease or disorder as described above, by administering tothe subject a therapeutic agent (e.g., an RNAi agent or vector ortransgene encoding same). Subjects at risk for the disease can beidentified by, for example, any or a combination of diagnostic orprognostic assays as described herein. Administration of a prophylacticagent can occur prior to the manifestation of symptoms characteristic ofthe disease or disorder, such that the disease or disorder is preventedor, alternatively, delayed in its progression.

Another aspect of the invention pertains to methods treating subjectstherapeutically, i.e., alter onset of symptoms of the disease ordisorder.

With regards to both prophylactic and therapeutic methods of treatment,such treatments may be specifically tailored or modified, based onknowledge obtained from the field of pharmacogenomics.“Pharmacogenomics,” as used herein, refers to the application ofgenomics technologies such as gene sequencing, statistical genetics, andgene expression analysis to drugs in clinical development and on themarket. More specifically, the term refers the study of how a patient'sgenes determine his or her response to a drug (e.g., a patient's “drugresponse phenotype,” or “drug response genotype”). Thus, another aspectof the invention provides methods for tailoring an individual'sprophylactic or therapeutic treatment with either the target genemolecules of the present invention or target gene modulators accordingto that individual's drug response genotype. Pharmacogenomics allows aclinician or physician to target prophylactic or therapeutic treatmentsto patients who will most benefit from the treatment and to avoidtreatment of patients who will experience toxic drug-related sideeffects.

Therapeutic agents can be tested in an appropriate animal model. Forexample, an RNAi agent (or expression vector or transgene encoding same)as described herein can be used in an animal model to determine theefficacy, toxicity, or side effects of treatment with said agent.Alternatively, a therapeutic agent can be used in an animal model todetermine the mechanism of action of such an agent. For example, anagent can be used in an animal model to determine the efficacy,toxicity, or side effects of treatment with such an agent.Alternatively, an agent can be used in an animal model to determine themechanism of action of such an agent.

A pharmaceutical composition containing an RNA silencing agent of theinvention can be administered to any patient diagnosed as having or atrisk for developing a neurodegenerative disease. In one embodiment, thepatient is diagnosed as having a neurological disorder, and the patientis otherwise in general good health. For example, the patient is notterminally ill, and the patient is likely to live at least 2, 3, 5 ormore years following diagnosis. The patient can be treated immediatelyfollowing diagnosis, or treatment can be delayed until the patient isexperiencing more debilitating symptoms, such as motor fluctuations anddyskinesis in Parkinson's disease patients. In another embodiment, thepatient has not reached an advanced stage of the disease.

An RNA silencing agent modified for enhanced uptake into neural cellscan be administered at a unit dose less than about 1.4 mg per kg ofbodyweight, or less than 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005,0.001, 0.0005, 0.0001, 0.00005 or 0.00001 mg per kg of bodyweight, andless than 200 nmole of RNA agent (e.g., about 4.4×10¹⁶ copies) per kg ofbodyweight, or less than 1500, 750, 300, 150, 75, 15, 7.5, 1.5, 0.75,0.15, 0.075, 0.015, 0.0075, 0.0015, 0.00075, 0.00015 nmole of RNAsilencing agent per kg of bodyweight. The unit dose, for example, can beadministered by injection (e.g., intravenous or intramuscular,intrathecally, or directly into the brain), an inhaled dose, or atopical application. Particularly preferred dosages are less than 2, 1,or 0.1 mg/kg of body weight.

Delivery of an RNA silencing agent directly to an organ (e.g., directlyto the brain) can be at a dosage on the order of about 0.00001 mg toabout 3 mg per organ, or preferably about 0.0001-0.001 mg per organ,about 0.03-3.0 mg per organ, about 0.1-3.0 mg per eye or about 0.3-3.0mg per organ. The dosage can be an amount effective to treat or preventa neurodegenerative disease or disorder, e.g., AD or ALS. In oneembodiment, the unit dose is administered less frequently than once aday, e.g., less than every 2, 4, 8 or 30 days. In another embodiment,the unit dose is not administered with a frequency (e.g., not a regularfrequency). For example, the unit dose may be administered a singletime. In one embodiment, the effective dose is administered with othertraditional therapeutic modalities.

In one embodiment, a subject is administered an initial dose, and one ormore maintenance doses of an RNA silencing agent. The maintenance doseor doses are generally lower than the initial dose, e.g., one-half lessof the initial dose. A maintenance regimen can include treating thesubject with a dose or doses ranging from 0.01 g to 1.4 mg/kg of bodyweight per day, e.g., 10, 1, 0.1, 0.01, 0.001, or 0.00001 mg per kg ofbodyweight per day. The maintenance doses are preferably administered nomore than once every 5, 10, or 30 days. Further, the treatment regimenmay last for a period of time which will vary depending upon the natureof the particular disease, its severity and the overall condition of thepatient. In preferred embodiments the dosage may be delivered no morethan once per day, e.g., no more than once per 24, 36, 48, or morehours, e.g., no more than once every 5 or 8 days. Following treatment,the patient can be monitored for changes in his condition and foralleviation of the symptoms of the disease state. The dosage of thecompound may either be increased in the event the patient does notrespond significantly to current dosage levels, or the dose may bedecreased if an alleviation of the symptoms of the disease state isobserved, if the disease state has been ablated, or if undesiredside-effects are observed.

The effective dose can be administered in a single dose or in two ormore doses, as desired or considered appropriate under the specificcircumstances. If desired to facilitate repeated or frequent infusions,implantation of a delivery device, e.g., a pump, semi-permanent stent(e.g., intravenous, intraperitoneal, intracisternal or intracapsular),or reservoir may be advisable. In one embodiment, a pharmaceuticalcomposition includes a plurality of RNA silencing agent species. Inanother embodiment, the RNA silencing agent species has sequences thatare non-overlapping and non-adjacent to another species with respect toa naturally occurring target sequence. In another embodiment, theplurality of RNA silencing agent species is specific for differentnaturally occurring target genes. In another embodiment, the RNAsilencing agent is allele specific. In another embodiment, the pluralityof RNA silencing agent species target two or more target sequences(e.g., two, three, four, five, six, or more target sequences).

Following successful treatment, it may be desirable to have the patientundergo maintenance therapy to prevent the recurrence of the diseasestate, wherein the compound of the invention is administered inmaintenance doses, ranging from 0.01 g to 100 g per kg of body weight(see U.S. Pat. No. 6,107,094).

The concentration of the RNA silencing agent composition is an amountsufficient to be effective in treating or preventing a disorder or toregulate a physiological condition in humans. The concentration oramount of RNA silencing agent administered will depend on the parametersdetermined for the agent and the method of administration, e.g. nasal,buccal, or pulmonary. For example, nasal formulations tend to requiremuch lower concentrations of some ingredients in order to avoidirritation or burning of the nasal passages. It is sometimes desirableto dilute an oral formulation up to 10-100 times in order to provide asuitable nasal formulation.

Certain factors may influence the dosage required to effectively treat asubject, including but not limited to the severity of the disease ordisorder, previous treatments, the general health and/or age of thesubject, and other diseases present. Moreover, treatment of a subjectwith a therapeutically effective amount of an RNA silencing agent caninclude a single treatment or, preferably, can include a series oftreatments. It will also be appreciated that the effective dosage of anRNA silencing agent for treatment may increase or decrease over thecourse of a particular treatment. Changes in dosage may result andbecome apparent from the results of diagnostic assays as describedherein. For example, the subject can be monitored after administering anRNA silencing agent composition. Based on information from themonitoring, an additional amount of the RNA silencing agent compositioncan be administered.

Dosing is dependent on severity and responsiveness of the diseasecondition to be treated, with the course of treatment lasting fromseveral days to several months, or until a cure is effected or adiminution of disease state is achieved. Optimal dosing schedules can becalculated from measurements of drug accumulation in the body of thepatient. Persons of ordinary skill can easily determine optimum dosages,dosing methodologies and repetition rates. Optimum dosages may varydepending on the relative potency of individual compounds, and cangenerally be estimated based on EC50s found to be effective in in vitroand in vivo animal models. In some embodiments, the animal modelsinclude transgenic animals that express a human gene, e.g., a gene thatproduces a target RNA, e.g., an RNA expressed in a neural cell. Thetransgenic animal can be deficient for the corresponding endogenous RNA.In another embodiment, the composition for testing includes an RNAsilencing agent that is complementary, at least in an internal region,to a sequence that is conserved between the target RNA in the animalmodel and the target RNA in a human.

IX. Pharmaceutical Compositions and Methods of Administration

The invention pertains to uses of the above-described agents forprophylactic and/or therapeutic treatments as described infra.Accordingly, the modulators (e.g., branched oligonucleotides comprisingRNA silencing agents) of the present application can be incorporatedinto pharmaceutical compositions suitable for administration. Suchcompositions typically comprise the nucleic acid molecule, protein,antibody, or branched oligonucleotide compound and a pharmaceuticallyacceptable carrier. As used herein, the language “pharmaceuticallyacceptable carrier” is intended to include any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, intraperitoneal, intramuscular, oral (e.g., inhalation),transdermal (topical), and transmucosal administration. In certainexemplary embodiments, a pharmaceutical composition of the invention isdelivered to the cerebrospinal fluid (CSF) by a route of administrationthat includes, but is not limited to, intrastriatal (IS) administration,intracerebroventricular (ICV) administration and intrathecal (IT)administration (e.g., via a pump, an infusion or the like). Solutions orsuspensions used for parenteral, intradermal, or subcutaneousapplication can include the following components: a sterile diluent suchas water for injection, saline solution, fixed oils, polyethyleneglycols, glycerine, propylene glycol or other synthetic solvents;antibacterial agents such as benzyl alcohol or methyl parabens;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. pH can be adjusted with acids or bases,such as hydrochloric acid or sodium hydroxide. The parenteralpreparation can be enclosed in ampoules, disposable syringes or multipledose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous, IS, ICV and/or ITadministration, suitable carriers include physiological saline,bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) orphosphate buffered saline (PBS). In all cases, the composition must besterile and should be fluid to the extent that easy syringabilityexists. It must be stable under the conditions of manufacture andstorage and must be preserved against the contaminating action ofmicroorganisms such as bacteria and fungi. The carrier can be a solventor dispersion medium containing, for example, water, ethanol, polyol(for example, glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), and suitable mixtures thereof. The properfluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent which delays absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

The RNA silencing agents can also be administered by transfection orinfection using methods known in the art, including but not limited tothe methods described in McCaffrey et al. (2002), Nature, 418(6893),38-9 (hydrodynamic transfection); Xia et al. (2002), Nature Biotechnol.,20(10), 1006-10 (viral-mediated delivery); or Putnam (1996), Am. J.Health Syst. Pharm. 53(2), 151-160, erratum at Am. J. Health Syst.Pharm. 53(3), 325 (1996).

The RNA silencing agents can also be administered by any method suitablefor administration of nucleic acid agents, such as a DNA vaccine. Thesemethods include gene guns, bio injectors, and skin patches as well asneedle-free methods such as the micro-particle DNA vaccine technologydisclosed in U.S. Pat. No. 6,194,389, and the mammalian transdermalneedle-free vaccination with powder-form vaccine as disclosed in U.S.Pat. No. 6,168,587. Additionally, intranasal delivery is possible, asdescribed in, inter alia, Hamajima et al. (1998), Clin. Immunol.Immunopathol., 88(2),205-10. Liposomes (e.g., as described in U.S. Pat.No. 6,472,375) and microencapsulation can also be used. Biodegradabletargetable microparticle delivery systems can also be used (e.g., asdescribed in U.S. Pat. No. 6,471,996).

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds that exhibit large therapeutic indices are preferred. Althoughcompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the EC50 (i.e., the concentration ofthe test compound which achieves a half-maximal response) as determinedin cell culture. Such information can be used to more accuratelydetermine useful doses in humans. Levels in plasma may be measured, forexample, by high performance liquid chromatography.

The pharmaceutical compositions can be included in a container, pack ordispenser together with optional instructions for administration.

As defined herein, a therapeutically effective amount of a RNA silencingagent (i.e., an effective dosage) depends on the RNA silencing agentselected. For instance, if a plasmid encoding shRNA is selected, singledose amounts in the range of approximately 1 g to 1000 mg may beadministered; in some embodiments, 10, 30, 100 or 1000 g may beadministered. In some embodiments, 1-5 g of the compositions can beadministered. The compositions can be administered one from one or moretimes per day to one or more times per week; including once every otherday. The skilled artisan will appreciate that certain factors mayinfluence the dosage and timing required to effectively treat a subject,including but not limited to the severity of the disease or disorder,previous treatments, the general health and/or age of the subject, andother diseases present. Moreover, treatment of a subject with atherapeutically effective amount of a protein, polypeptide, or antibodycan include a single treatment or, preferably, can include a series oftreatments.

The nucleic acid molecules of the invention can be inserted intoexpression constructs, e.g., viral vectors, retroviral vectors,expression cassettes, or plasmid viral vectors, e.g., using methodsknown in the art, including but not limited to those described in Xia etal., (2002), Supra. Expression constructs can be delivered to a subjectby, for example, inhalation, orally, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. (1994), Proc. Natl. Acad. Sci. USA, 91,3054-3057). The pharmaceutical preparation of the delivery vector caninclude the vector in an acceptable diluent, or can comprise a slowrelease matrix in which the delivery vehicle is imbedded. Alternatively,where the complete delivery vector can be produced intact fromrecombinant cells, e.g., retroviral vectors, the pharmaceuticalpreparation can include one or more cells which produce the genedelivery system.

The nucleic acid molecules of the invention can also include smallhairpin RNAs (shRNAs), and expression constructs engineered to expressshRNAs. Transcription of shRNAs is initiated at a polymerase III (polIII) promoter, and is thought to be terminated at position 2 of a4-5-thymine transcription termination site. Upon expression, shRNAs arethought to fold into a stem-loop structure with 3′ UU-overhangs;subsequently, the ends of these shRNAs are processed, converting theshRNAs into siRNA-like molecules of about 21 nucleotides. Brummelkamp etal. (2002), Science, 296, 550-553; Lee et al, (2002). supra; Miyagishiand Taira (2002), Nature Biotechnol., 20, 497-500; Paddison et al.(2002), supra; Paul (2002), supra; Sui (2002) supra; Yu et al. (2002),supra.

The expression constructs may be any construct suitable for use in theappropriate expression system and include, but are not limited toretroviral vectors, linear expression cassettes, plasmids and viral orvirally-derived vectors, as known in the art. Such expression constructsmay include one or more inducible promoters, RNA Pol III promotersystems such as U6 snRNA promoters or H1 RNA polymerase III promoters,or other promoters known in the art. The constructs can include one orboth strands of the siRNA. Expression constructs expressing both strandscan also include loop structures linking both strands, or each strandcan be separately transcribed from separate promoters within the sameconstruct. Each strand can also be transcribed from a separateexpression construct, Tuschl (2002), Supra.

In certain exemplary embodiments, a composition that includes an RNAsilencing agent of the invention can be delivered to the nervous systemof a subject by a variety of routes. Exemplary routes includeintrathecal, parenchymal (e.g., in the brain), nasal, and oculardelivery. The composition can also be delivered systemically, e.g., byintravenous, subcutaneous or intramuscular injection, which isparticularly useful for delivery of the RNA silencing agents toperipheral neurons. A preferred route of delivery is directly to thebrain, e.g., into the ventricles or the hypothalamus of the brain, orinto the lateral or dorsal areas of the brain. The RNA silencing agentsfor neural cell delivery can be incorporated into pharmaceuticalcompositions suitable for administration.

For example, compositions can include one or more species of an RNAsilencing agent and a pharmaceutically acceptable carrier. Thepharmaceutical compositions of the present invention may be administeredin a number of ways depending upon whether local or systemic treatmentis desired and upon the area to be treated. Administration may betopical (including ophthalmic, intranasal, transdermal), oral orparenteral. Parenteral administration includes intravenous drip,subcutaneous, intraperitoneal or intramuscular injection, intrathecal,or intraventricular (e.g., intracerebroventricular) administration. Incertain exemplary embodiments, an RNA silencing agent of the inventionis delivered across the Blood-Brain-Barrier (BBB) suing a variety ofsuitable compositions and methods described herein.

The route of delivery can be dependent on the disorder of the patient.For example, a subject diagnosed with a neurodegenerative disease can beadministered an RNA silencing agent of the invention directly into thebrain (e.g., into the globus pallidus or the corpus striatum of thebasal ganglia, and near the medium spiny neurons of the corpusstriatum). In addition to an RNA silencing agent of the invention, apatient can be administered a second therapy, e.g., a palliative therapyand/or disease-specific therapy. The secondary therapy can be, forexample, symptomatic (e.g., for alleviating symptoms), neuroprotective(e.g., for slowing or halting disease progression), or restorative(e.g., for reversing the disease process). Other therapies can includepsychotherapy, physiotherapy, speech therapy, communicative and memoryaids, social support services, and dietary advice.

An RNA silencing agent can be delivered to neural cells of the brain.Delivery methods that do not require passage of the composition acrossthe blood-brain barrier can be utilized. For example, a pharmaceuticalcomposition containing an RNA silencing agent can be delivered to thepatient by injection directly into the area containing thedisease-affected cells. For example, the pharmaceutical composition canbe delivered by injection directly into the brain. The injection can beby stereotactic injection into a particular region of the brain (e.g.,the substantia nigra, cortex, hippocampus, striatum, or globuspallidus). The RNA silencing agent can be delivered into multipleregions of the central nervous system (e.g., into multiple regions ofthe brain, and/or into the spinal cord). The RNA silencing agent can bedelivered into diffuse regions of the brain (e.g., diffuse delivery tothe cortex of the brain).

In one embodiment, the RNA silencing agent can be delivered by way of acannula or other delivery device having one end implanted in a tissue,e.g., the brain, e.g., the substantia nigra, cortex, hippocampus,striatum or globus pallidus of the brain. The cannula can be connectedto a reservoir of RNA silencing agent. The flow or delivery can bemediated by a pump, e.g., an osmotic pump or minipump, such as an Alzetpump (Durect, Cupertino, Calif.). In one embodiment, a pump andreservoir are implanted in an area distant from the tissue, e.g., in theabdomen, and delivery is effected by a conduit leading from the pump orreservoir to the site of release. Devices for delivery to the brain aredescribed, for example, in U.S. Pat. Nos. 6,093,180, and 5,814,014.

An RNA silencing agent of the invention can be further modified suchthat it is capable of traversing the blood brain barrier. For example,the RNA silencing agent can be conjugated to a molecule that enables theagent to traverse the barrier. Such modified RNA silencing agents can beadministered by any desired method, such as by intraventricular orintramuscular injection, or by pulmonary delivery, for example.

In some embodiments, exosomes are used to deliver an RNA silencing agentof the invention. Exosomes can cross the BBB and deliver siRNAs,antisense oligonucleotides, chemotherapeutic agents and proteinsspecifically to neurons after systemic injection (See, Alvarez-Erviti L,Seow Y, Yin H, Betts C, Lakhal S, Wood M J. (2011). Delivery of siRNA tothe mouse brain by systemic injection of targeted exosomes. NatBiotechnol. 2011 April; 29(4):341-5. doi: 10.1038/nbt.1807;El-Andaloussi S, Lee Y, Lakhal-Littleton S, Li J, Seow Y, Gardiner C,Alvarez-Erviti L, Sargent I L, Wood M J. (2011). Exosome-mediateddelivery of siRNA in vitro and in vivo. Nat Protoc. 2012 December;7(12):2112-26. doi: 10.1038/nprot.2012.131; E L Andaloussi S, Mager I,Breakefield X O, Wood M J. (2013). Extracellular vesicles: biology andemerging therapeutic opportunities. Nat Rev Drug Discov. 2013 May;12(5):347-57. doi: 10.1038/nrd3978; El Andaloussi S, Lakhal S, Mager I,Wood M J. (2013). Exosomes for targeted siRNA delivery across biologicalbarriers. Adv Drug Deliv Rev. 2013 March; 65(3):391-7. doi:10.1016/j.addr.2012.08.008).

In some embodiments, one or more lipophilic molecules are used to allowdelivery of an RNA silencing agent of the invention past the BBB(Alvarez-Ervit (2011)). The RNA silencing agent would then be activated,e.g., by enzyme degradation of the lipophilic disguise to release thedrug into its active form.

In some embodiments, one or more receptor-mediated permeabilizingcompounds can be used to increase the permeability of the BBB to allowdelivery of an RNA silencing agent of the invention. These drugsincrease the permeability of the BBB temporarily by increasing theosmotic pressure in the blood which loosens the tight junctions betweenthe endothelial cells ((El-Andaloussi (2012)). By loosening the tightjunctions normal intravenous injection of an RNA silencing agent can beperformed.

In some embodiments, nanoparticle-based delivery systems are used todeliver an RNA silencing agent of the invention across the BBB. As usedherein, “nanoparticles” refer to polymeric nanoparticles that aretypically solid, biodegradable, colloidal systems that have been widelyinvestigated as drug or gene carriers (S. P. Egusquiaguirre, M. Igartua,R. M. Hernandez, and J. L. Pedraz, “Nanoparticle delivery systems forcancer therapy: advances in clinical and preclinical research,” Clinicaland Translational Oncology, vol. 14, no. 2, pp. 83-93, 2012). Polymericnanoparticles are classified into two major categories, natural polymersand synthetic polymers. Natural polymers for siRNA delivery include, butare not limited to, cyclodextrin, chitosan, and atelocollagen (Y. Wang,Z. Li, Y. Han, L. H. Liang, and A. Ji, “Nanoparticle-based deliverysystem for application of siRNA in vivo,” Current Drug Metabolism, vol.11, no. 2, pp. 182-196, 2010). Synthetic polymers include, but are notlimited to, polyethyleneimine (PEI), poly(dl-lactide-co-glycolide)(PLGA), and dendrimers, which have been intensively investigated (X.Yuan, S. Naguib, and Z. Wu, “Recent advances of siRNA delivery bynanoparticles,” Expert Opinion on Drug Delivery, vol. 8, no. 4, pp.521-536, 2011). For a review of nanoparticles and other suitabledelivery systems, See Jong-Min Lee, Tae-Jong Yoon, and Young-Seok Cho,“Recent Developments in Nanoparticle-Based siRNA Delivery for CancerTherapy,” BioMed Research International, vol. 2013, Article ID 782041,10 pages, 2013. doi:10.1155/2013/782041 (incorporated by reference inits entirety.)

An RNA silencing agent of the invention can be administered ocularly,such as to treat retinal disorder, e.g., a retinopathy. For example, thepharmaceutical compositions can be applied to the surface of the eye ornearby tissue, e.g., the inside of the eyelid. They can be appliedtopically, e.g., by spraying, in drops, as an eyewash, or an ointment.Ointments or droppable liquids may be delivered by ocular deliverysystems known in the art such as applicators or eye droppers. Suchcompositions can include mucomimetics such as hyaluronic acid,chondroitin sulfate, hydroxypropyl methylcellulose or poly(vinylalcohol), preservatives such as sorbic acid, EDTA or benzylchroniumchloride, and the usual quantities of diluents and/or carriers. Thepharmaceutical composition can also be administered to the interior ofthe eye, and can be introduced by a needle or other delivery devicewhich can introduce it to a selected area or structure. The compositioncontaining the RNA silencing agent can also be applied via an ocularpatch.

In general, an RNA silencing agent of the invention can be administeredby any suitable method. As used herein, topical delivery can refer tothe direct application of an RNA silencing agent to any surface of thebody, including the eye, a mucous membrane, surfaces of a body cavity,or to any internal surface. Formulations for topical administration mayinclude transdermal patches, ointments, lotions, creams, gels, drops,sprays, and liquids. Conventional pharmaceutical carriers, aqueous,powder or oily bases, thickeners and the like may be necessary ordesirable. Topical administration can also be used as a means toselectively deliver the RNA silencing agent to the epidermis or dermisof a subject, or to specific strata thereof, or to an underlying tissue.

Compositions for intrathecal or intraventricular (e.g.,intracerebroventricular) administration may include sterile aqueoussolutions which may also contain buffers, diluents and other suitableadditives. Compositions for intrathecal or intraventricularadministration preferably do not include a transfection reagent or anadditional lipophilic moiety besides, for example, the lipophilic moietyattached to the RNA silencing agent.

Formulations for parenteral administration may include sterile aqueoussolutions which may also contain buffers, diluents and other suitableadditives. Intraventricular injection may be facilitated by anintraventricular catheter, for example, attached to a reservoir. Forintravenous use, the total concentration of solutes should be controlledto render the preparation isotonic.

An RNA silencing agent of the invention can be administered to a subjectby pulmonary delivery. Pulmonary delivery compositions can be deliveredby inhalation of a dispersion so that the composition within thedispersion can reach the lung where it can be readily absorbed throughthe alveolar region directly into blood circulation. Pulmonary deliverycan be effective both for systemic delivery and for localized deliveryto treat diseases of the lungs. In one embodiment, an RNA silencingagent administered by pulmonary delivery has been modified such that itis capable of traversing the blood brain barrier.

Pulmonary delivery can be achieved by different approaches, includingthe use of nebulized, aerosolized, micellular and dry powder-basedformulations. Delivery can be achieved with liquid nebulizers,aerosol-based inhalers, and dry powder dispersion devices. Metered-dosedevices are preferred. One of the benefits of using an atomizer orinhaler is that the potential for contamination is minimized because thedevices are self-contained. Dry powder dispersion devices, for example,deliver drugs that may be readily formulated as dry powders. An RNAsilencing agent composition may be stably stored as lyophilized orspray-dried powders by itself or in combination with suitable powdercarriers. The delivery of a composition for inhalation can be mediatedby a dosing timing element which can include a timer, a dose counter,time measuring device, or a time indicator which when incorporated intothe device enables dose tracking, compliance monitoring, and/or dosetriggering to a patient during administration of the aerosol medicament.

The types of pharmaceutical excipients that are useful as carriersinclude stabilizers such as human serum albumin (HSA), bulking agentssuch as carbohydrates, amino acids and polypeptides; pH adjusters orbuffers; salts such as sodium chloride; and the like. These carriers maybe in a crystalline or amorphous form or may be a mixture of the two.

Bulking agents that are particularly valuable include compatiblecarbohydrates, polypeptides, amino acids or combinations thereof.Suitable carbohydrates include monosaccharides such as galactose,D-mannose, sorbose, and the like; disaccharides, such as lactose,trehalose, and the like; cyclodextrins, such as2-hydroxypropyl-beta-cyclodextrin; and polysaccharides, such asraffinose, maltodextrins, dextrans, and the like; alditols, such asmannitol, xylitol, and the like. A preferred group of carbohydratesincludes lactose, trehalose, raffinose maltodextrins, and mannitol.Suitable polypeptides include aspartame. Amino acids include alanine andglycine, with glycine being preferred.

Suitable pH adjusters or buffers include organic salts prepared fromorganic acids and bases, such as sodium citrate, sodium ascorbate, andthe like; sodium citrate is preferred.

An RNA silencing agent of the invention can be administered by oral andnasal delivery. For example, drugs administered through these membraneshave a rapid onset of action, provide therapeutic plasma levels, avoidfirst pass effect of hepatic metabolism, and avoid exposure of the drugto the hostile gastrointestinal (GI) environment. Additional advantagesinclude easy access to the membrane sites so that the drug can beapplied, localized and removed easily. In one embodiment, an RNAsilencing agent administered by oral or nasal delivery has been modifiedto be capable of traversing the blood-brain barrier.

In one embodiment, unit doses or measured doses of a composition thatinclude RNA silencing agents are dispensed by an implanted device. Thedevice can include a sensor that monitors a parameter within a subject.For example, the device can include a pump, such as an osmotic pump and,optionally, associated electronics.

An RNA silencing agent can be packaged in a viral natural capsid or in achemically or enzymatically produced artificial capsid or structurederived therefrom.

X. Kits

In certain other aspects, the invention provides kits that include asuitable container comprising a modified siRNA, said modified siRNAhaving a 5′ end, a 3′ end, that is complementary to a target, whereinthe siRNA comprises a sense and antisense strand, and at least onemodified intersubunit linkage of Formula (I) as described supra. Kitsmay also comprise a pharmaceutical formulation of an RNA silencingagent, e.g., a double-stranded RNA silencing agent, or sRNA agent,(e.g., a precursor, e.g., a larger RNA silencing agent which can beprocessed into a sRNA agent, or a DNA which encodes an RNA silencingagent, e.g., a double-stranded RNA silencing agent, or sRNA agent, orprecursor thereof). In some embodiments the individual components of thepharmaceutical formulation may be provided in one container.Alternatively, it may be desirable to provide the components of thepharmaceutical formulation separately in two or more containers, e.g.,one container for an RNA silencing agent preparation, and at leastanother for a carrier compound. The kit may be packaged in a number ofdifferent configurations such as one or more containers in a single box.The different components can be combined, e.g., according toinstructions provided with the kit. The components can be combinedaccording to a method described herein, e.g., to prepare and administera pharmaceutical composition. The kit can also include a deliverydevice.

It will be readily apparent to those skilled in the art that othersuitable modifications and adaptations of the methods described hereinmay be made using suitable equivalents without departing from the scopeof the embodiments disclosed herein. Having now described someembodiments in detail, the same will be more clearly understood byreference to the following example, which is included for purposes ofillustration only and is not intended to be limiting.

EXAMPLES Example 1. Synthesis of a Phosphinate-Modified IntersubunitLinkage

A method for preparing a phosphinate-modified intersubunit linkage ofthe invention is summarized in FIGS. 2A-2C. This method involves Jonesoxidation from a free alcohol to the corresponding ketone followed by aWittig olefination to achieve the exomethylene moiety shown inintermediate compound 3. Protecting of the amide with BOM followed byhydroboration-oxidation results in the free alcohol intermediate 5.Mesylation followed by a modified Finkelstein reaction produces theiodinated intermediate 7, which then undergoes further functionalizationto achieve the methyl phosphinate monomer 9.

To achieve monomer 18, various protection and deprotection steps areemployed to achieve intermediate 13. IBX oxidation produces thecorresponding ketone followed by Wittig olefination to access themethylene. Once again, hydroboration-oxidation followed by mesylationand Finkelstein reaction results in monomer 18.

Combining monomers 9 and 18 under basic conditions producesphosphinate-linked dimer 19. Acid-mediated and Pearlman's catalyzeddeprotection followed by further phosphanamine functionalization resultsin dimer 22.

Example 2. Synthesis of a Phosphonate-Modified Intersubunit Linkage

A method for preparing a phosphonate-modified intersubunit linkage ofthe invention is summarized in FIGS. 3A and 3B.

Amide protection with BOM followed by methoxyphosphanaminefunctionalization results in intermediate 24. Reduction using tetrazoleand water were employed to achieve monomer 25.

Monomers 18 and 25 were combined under basic conditions to producephosphonate dimer 26. Acid-mediated and Pearlman's catalyzeddeprotection followed by further phosphanamine functionalization resultsin dimer 29.

Example 3. Solid Support Oligonucleotide Extension

The method of assembling modified oligonucleotides having phosphinateand phosphonate intersubunit linkages is summarized in FIG. 4. Theoligonucleotides provided herein can be assembled using a solid supportmechanism wherein the modified intersubunit linkages can be selectivelyinserted into oligonucleotide sequences.

Example 4. Synthesis Vinyl Phosphonate-Modified Intersubunit Linkages

A synthetic approach for preparing vinyl phosphonate modifiedintersubunit linkages of the invention is summarized in FIG. 5.

Synthesis of Compound 2a

TBDPS protection of the secondary alcohol on compound 1a was performedunder standard conditions well known in the art of organic synthesis toproduce compound 2a.

Synthesis of Compound 3a

Anhydrous solution of compound 2a (16.6 g, 20.8 mmol) in pyridine (100mL) was added anhydrous DIPEA (6.5 mL, 37.4 mmol) and benzoyl chloride(3.6 mL, 31.2 mmol). After the mixture was stirred for 4 h at rt, excesspyridine was evaporated and diluted with CH₂Cl₂. The organic solutionwas washed by sat. aq. NaHCO₃. The organic layer was collected, driedover MgSO₄, filtered and evaporated. Obtained crude material waspurified by silica gel column chromatography (hexane-ethyl acetate, 4:1to 1:1) yielding compound 3a as a slightly yellow foam (14.5 g, 78%); ¹HNMR (500 MHz, CDCl₃) δ 7.88-7.87 (m, 2H), 7.84 (d, 1H, J=8.3 Hz),7.67-7.58 (m, 5H), 7.48-7.45 (m, 4H), 7.39-7.32 (m, 4H), 7.25-7.23 (m,3H), 7.18-7.17 (m, 2H), 7.12-7.07 (m, 4H), 6.80-6.75 (m, 4H), 6.08 (dd,1H, J_(HH)=1.5 Hz, J_(HF)=15.2 Hz), 5.14, (d, 1H, J_(HH)=8.3 Hz), 4.59(ddd, 1H, J_(HH)=3.7, 1.5 Hz, J_(HF)=51.9 Hz), 4.43 (ddd, 1H,J_(HH)=7.4, 4.0 Hz, J_(HF)=19.1 Hz), 4.24-4.23 (m, 1H), 3.79 (s, 6H),3.62 (dd, 1H, J_(HH)=11.2, 2.0 Hz), 3.35 (dd, 1H, J_(HH)=11.1, 2.0 Hz),1.00 (s, 9H); ¹³C NMR (126 Hz, CDCl₃) δ 168.4, 161.8, 158.72, 158.66,148.9, 143.9, 139.4, 135.71, 135.70, 135.1, 134.8, 134.7, 132.3, 132.2,131.3, 130.4, 130.2, 130.1, 129.1, 128.2, 128.0, 127.91, 127.89, 127.2,113.19, 113.16, 102.2, 92.5 (d, JCF=194.4 Hz), 87.7 (d, J_(CF)=34.5 Hz),87.2, 82.4, 70.0 (d, J_(CF)=15.4 Hz), 60.7, 60.4, 55.2, 26.6.

Synthesis of Compound 4a

Compound 3a (14.5 g, 16.3 mmol) was dissolved into 3% trichloroaceticacid/CH₂Cl₂ solution (200 mL) containing triethylsilane (8.0 mL, 50.1mmol) and stirred for 1 h at rt. After the solution was washed by sat.aq. NaHCO₃ three times, collected organic layer was dried over MgSO₄,filtered, and evaporated. Obtained crude material was purified by silicagel column chromatography (hexane/ethyl acetate, 4:1 to 3:7) yieldingcompound 4a as a white foam (8.67 g, 91%); ¹H NMR (500 MHz, CDCl₃) δ7.89-7.88 (m, 2H), 7.68-7.64 (6H, m), 7.51-7.45 (m, 4H), 7.42-7.38 (4H,m), 5.93 (dd, 1H, J_(HH)=2.9 Hz, J_(HF)=15.1 Hz), 5.73 (d, 1H,J_(HH)=8.2 Hz), 4.74 (ddd, 1H, J_(HH)=4.1, 3.2 Hz, J_(HF)=52.2 Hz), 4.31(ddd, 1H, J_(HH)=5.8, 4.7, J_(HF)=15.4 Hz), 4.11-4.09 (m, 1H), 3.82-3.79(m, 1H), 3.39 (ddd, 1H, J_(HH)=12.1, 5.6, 1.5 Hz), 1.64 (br, 1H), 1.11(s, 9H); ¹³C NMR (126 Hz, CDCl₃) δ 168.3, 161.8, 149.0, 140.5, 135.7,135.2, 132.8, 132.3, 131.3, 130.5, 130.4, 130.3, 129.2, 128.02, 127.96,102.4, 91.8 (d, J_(CF)=91.8 Hz), 89.5 (d, J_(CF)=33.6 Hz), 69.5 (d,J_(CF)=69.5 Hz), 60.3, 26.8.

Synthesis of Compound 6a

Anhydrous solution of compound 4a (6.5 g, 11.0 mmol) was added IBX (7.7g, 27.6 mmol) and stirred for 2 h at 85° C. After cooling the mixture inan ice bath, the precipitate in the solution was filtered off throughcelite. Collected eluent was evaporated, co-evaporated with anhydrousCH₃CN three times under argon atmosphere, and obtained compound 5a as awhite foam was used without further purification. In a separate flask,anhydrous CH₂Cl₂ (25 mL) solution containing CBr₄ (7.3 g, 22.1 mmol) wasadded PPh₃ (11.6 g, 44.2 mmol) at 0° C. and stirred for 0.5 h at 0° C.To this solution, anhydrous CH₂Cl₂ solution (25 mL) of compound 5a wasadded dropwise (10 min) at 0° C. and stirred for 2 h at 0° C. Afterdiluting with CH₂Cl₂, the organic solution was washed by aq. sat. NH₄Cl,dried over MgSO₄, filtered, and evaporated. Obtained material wasdissolved into minimum amount of diethyl ether and added dropwise toexcess diethyl ether solution under vigorously stirring at 0° C.Precipitate in solution was filtered off through celite and eluents wasevaporated. Obtained crude material was purified by silica gel columnchromatography (hexane/ethyl acetate, 9:1 to 1:1) yielding compound 6aas a white foam (4.3 g, 52%). 1H NMR (500 MHz, CDCl₃) δ 7.68-7.84 (m,2H), 7.70-7.65 (m, 3H), 7.60-7.58 (m, 2H), 7.52-7.49 (m, 2H), 7.42-7.36(m, 4H), 7.31-7.28 (m, 2H), 7.09 (d, 1H, J=8.2 Hz), 6.25 (d, 1H, J=8.9Hz), 5.75 (dd, 1H, J_(HF)=8.24 Hz), 5.49 (dd, 1H, J_(HF)=21.4 Hz), 4.77(t, 1H, J_(HH)=8.5 Hz, J_(HF)=8.5 Hz), 4.38 (dd, 1H, J_(HH)=4.1 Hz,J_(HF)=52.1 Hz), 4.25 (ddd, 1H, J_(HH)=8.1, 4.9 Hz, J_(HF)=19.4 Hz),1.10 (s, 9H); ¹³C NMR (126 Hz, CDCl₃) δ 167.9, 161.6, 148.3, 141.4,135.8, 134.7 (d, J_(C-Br)=139.0 Hz), 132.5, 132.2, 131.1, 130.5, 130.3,130.2, 129.2, 127.9, 102.7, 97.3, 93.3 (d, J_(CF)=39.1 Hz), 91.5 (d,J_(CF)=190.7 Hz), 82.4, 73.9 (d, J_(CF)=16.4 Hz), 26.7.

Synthesis of Compound 7a-E and 7a-Z

Anhydrous solution of compound 6a (4.2 g, 5.66 mmol) in DMF (25 mL) wasadded dimethylphosphite (2.09 mL, 22.6 mmol) and triethylamine (1.58 mL,11.3 mmol) at 0° C., and then stirred over night at rt. After thesolution was diluted with ethyl acetate, the organic solution was washedwith aq. sat. NH₄Cl and brine. Then the organic solution was dried overMgSO₄, filtered and evaporated. Obtained crude material was purifiedrepeatedly by silica gel column chromatography (hexane/ethyl acetate,9:1 to 1:1) until all pure isomeric compound were collected separately,giving compound 7a-E (1.95 g, 52%); ¹H NMR (500 MHz, CDCl₃) δ 7.87-7.85(m, 2H), 7.89-7.85 (m, 3H), 7.61-7.59 (m, 2H), 7.52-7.48 (m, 2H),7.45-7.32 (m, 6H), 7.08 (d, 1H, J_(HH)=8.2), 6.49 (d, 1H, J_(HH)=13.7),5.99 (dd, 1H, J_(HH)=13.7 Hz, 8.1 Hz), 5.75 (d, 1H, J_(HH)=8.2), 5.63(d, 1H, J_(HF)=19.8 Hz), 4.43 (dd, 1H, J_(HF)=52.6 Hz, J_(HH)=4.3 Hz),4.42 (t, 1H, J_(HH)=8.0 Hz), 4.07 (ddd, J_(HH)=7.8, 4.7 Hz, J_(HF)=19.5Hz), 1.08 (s, 9H); ¹³C NMR (126 Hz, CDCl₃) δ 148.4, 140.4, 135.8, 135.7,135.3, 133.3, 132.3, 132.4, 132.1, 131.1, 130.5, 130.4, 130.3, 129.2,127.95, 127.93, 112.4, 102.7, 91.7 (d, J_(CF)=36.3 Hz), 91.6 (d,J_(CF)=191.6 Hz), 82.8, 73.9 (d, J_(CF)=16.4 Hz), 26.7, 19.1; and 7a-Z(0.58 g, 15%); ¹H NMR (500 MHz, CDCl₃) δ 7.87-7.85 (m, 2H), 7.68-7.65(m, 3H), 7.61-7.59 (m, 2H), 7.52-7.48 (m, 2H), 7.42-7.39 (m, 2H),7.34-7.29 (m, 4H), 7.12 (d, 1H, J_(HH)=8.2 Hz), 6.51 (d, 1H, J_(HH)=7.4Hz), 5.96 (dd, 1H, J_(HH)=8.4 Hz, 7.4 Hz), 5.75 (d, 1H, J_(HH)=8.2 Hz),5.57 (dd, 1H, J_(HH)=1.2 Hz, J_(HF)=20.6 Hz), 5.04 (dd, 1H, J_(HH)=8.2Hz), 4.48 (J_(HH)=3.5 Hz, J_(HF)=53.1 Hz), 4.24 (ddd, 1H, J_(HH)=7.8,4.9 Hz, J_(HF)=18.6 Hz), 1.09 (s, 9H); ¹³C NMR (126 Hz, CDCl₃) δ 168.0,161.7, 148.4, 141.4, 135.9, 135.8, 135.2, 132.6, 132.5, 131.2, 130.6,130.5, 130.2, 130.1, 129.2, 127.8, 127.7, 114.5, 102.6, 93.0 (d,J_(CF)=37.2 Hz), 91.6 (d, J_(CF)=191.6 Hz), 80.3, 74.3 (d, J_(CF)=16.4Hz), 26.7, 19.1.

Synthesis of Compound 9a

Anhydrous compound 7a-E (1.95 g, 2.94 mmol) and Pd(OAc)₂ (125 mg, 0.59mmol) and [1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium (II)(652 mg, 1.18 mmol) were purged with argon, and then dissolved intoanhydrous THF (50 mL). After adding propylene oxide (2.06 mL, 29.4mmol), compound 8a (2.07 g, 3.24 mmol) was added in one portion andstirred at for 4 h at 70° C. After removing solvent under reducedpressure, the crude mixture was purified by silica gel columnchromatography (hexane/ethyl acetate, 50:50 to 0:100) and obtainedfractions containing compound 9a were further purified by silica gelcolumn chromatography (CH₂C2-MeOH, 0% to 5%) yielding compound 9a as amixture of diastereo-isomers (2.04 g, 57%); ³¹P NMR (202 MHz, CDCl₃) δ18.3.

Synthesis of Compound 10a

Compound 9a (2.0 g, 1.64 mmol) in anhydrous THF (22.5 mL) was added 1.0M TBAF-THF (2.5 mL, 2.5 mmol) and stirred at ambient temperature for 30min. After diluting with CH₂Cl₂ (120 mL), the organic layer was washedwith brine, dried over MgSO₄, filtered, and then evaporated. Obtainedcrude material was purified by silica gel column chromatography (1%TEA-CH₂C2/MeOH, 0% to 6%) yielding compound 10a (1.52 g, 94%); ³¹P NMR(202 MHz, CDCl₃) δ 19.0, 18.7.

Synthesis of Compound 11a

Compound 10a (589.7 mg, 0.6 mmol) was rendered anhydrous by repeatedco-evaporation with anhydrous CH₃CN and then dissolved into anhydrousCH₂Cl₂ (6.0 mL). To this solution N,N-diisopropylethylamine (0.31 mL,1.8 mmol) and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (0.16mL, 0.72 mmol) were added at 0° C. After stirring for 30 min at 0° C.,the reaction mixture was diluted with excess CH₂C2. The organic layerwas repeatedly washed with aq. sat. NaHCO₃, dried over MgSO₄, filtered,and evaporated. The obtained crude material was purified by silica gelcolumn chromatography (1% TEA-CH₂C2/MeOH, from 100% to 4%) yieldingcompound 11a as a white foam (570 mg, 80%); ³¹P NMR (202 MHz, CDCl₃) δ150.3, 151.2, 151.1, 151.0, 18.72, 18.65, 18.55, 18.3.

Synthesis of Compound 4b

Anhydrous solution of compound 3b (1.35 g, 2.0 mmol) in pyridine (10 mL)was added DIPEA (0.63 mL, 3.6 mmol) and benzoyl chloride (0.35 mL, 3.0mmol), and stirred for 3 h at rt. After diluting with excess CH₂C2, theorganic solution was washed with aq. sat. NaHCO₃ and brine. After dryingover MgSO₄, filtered and evaporating, obtained crude material was usedfor the next reaction without further purification. Obtained crudematerial containing compound 3b was added 3% trichloroacetic acid inCH₂Cl₂ (25 mL) and triethylsilane (1 mL, 6.26 mmol), and stirred for 1 hat rt. After the reaction mixture was diluted with CH₂C2, the solutionwas washed with sat. NaHCO₃aq. three times, dried over MgSO₄, filtered,then evaporated. Obtained crude material was purified by silica gelcolumn chromatography (hexane/ethyl acetate, 4:1 to 1:4) yielding purecompound 4b (596.7 mg, 63% in 2 steps); ¹H NMR (500 MHz, DMSO-d6) δ 8.13(d, 1H, J_(HH)=8.2 Hz), 7.95 (d, 2H, J_(HH)=7.3 Hz), 7.81 (t, 1H,J_(HH)=7.5 Hz), 7.69-7.68 (m, 2H), 7.64-7.59 (m, 4H), 7.49-7.42 (m, 6H),5.93 (d, 1H, J_(HH)=4.6 Hz), 5.26 (t, 1H, J_(HH)=4.6 Hz), 4.36 (dd, 1H,J_(HH)=4.6, 4.6 Hz), 4.02-4.00 (m, 1H), 3.65-3.61 (m, 1H), 3.54 (dd, 1H,J_(HH)=4.6, 4.6 Hz), 3.09 (s, 3H), 1.03 (s, 9H); ¹³C NMR (126 Hz,DMSO-d6) 169.8, 162.1, 149.5, 141.3, 136.1, 135.9, 135.8, 133.4, 133.2,131.5, 130.7, 130.52, 130.48, 130.0, 128.4, 128.3, 102.1, 86.7, 85.6,82.8, 79.7, 70.8, 60.2, 57.8, 27.2, 19.4; HRMS (ESI) m/z calcd forC33H35N207Si⁻ [M-H]⁻ m/z 599.2219, found m/z 599.2258.

Synthesis of compound 6b Anhydrous solution of compound 4b (300.4 mg,0.5 mmol) in CH₃CN (5 mL) was added IBX (350 mg, 1.3 mmol) and stirredfor 2 h at 85° C. After cooling the solution at 0° C., the precipitatewas filtered off by celite-filtration. Obtained eluent containingcompound 5b was evaporated, rendered anhydrous by repeatedco-evaporation with anhydrous CH₃CN, and used for the next reactionwithout further purification. Separatory prepared anhydrous solution ofCBr₄ (331.6 mg, 1.0 mmol) in CH₂Cl₂ (5.0 mL) was addedtriphenylphosphine (524.6 mg, 2.0 mmol) at 0° C. in one portion andstirred at 0° C. for 30 min. To this solution, compound 5b in anhydrousCH₂Cl₂ (1.5 mL) was added dropwise (10 min) at 0° C. and stirred for 2 hat 0° C. The solution was then diluted with CH₂C2 and washed with sat.NaHCO₃aq. and brine. After the organic solution was dried over MgSO₄,filtered and evaporated, obtained crude material was purified by silicagel column chromatography (hexane/ethyl acetate, 9:1 to 4:6) yieldingcompound 6b (210.9 mg, 56%); ¹H NMR (500 MHz, CDCl₃) δ 7.88 (d, 2H,J_(HH)=7.3 Hz), 7.70-7.62 (5H, m), 7.51-7.38 (m, 9H), 7.08 (d, 1H,J_(HH)=8.2 Hz), 6.26 (d, 1H, J_(HH)=8.6 Hz), 5.75 (d, 1H, J_(HH)=8.2Hz), 5.68 (d, 1H, J_(HH)=0.8 Hz), 4.84 (dd, 1H, J_(HH)=8.6 Hz, 8.6 Hz),3.86 (dd, 1H, J_(HH)=7.5 Hz, 5.0 Hz), 3.30 (s, 3H), 3.18 (br, 1H), 1.11(s, 9H); ¹³C NMR (126 Hz, CDCl₃) 168.3, 161.7, 148.6, 138.9, 135.9,135.8, 134.3, 132.6, 132.4, 131.2, 130.5, 130.4, 130.3, 129.2, 128.0,127.9, 102.4, 97.5, 90.0, 82.44, 82.39, 74.4, 58.2, 26.7, 19.1; HRMS(ESI) m/z calcd for C₃₄H₃₃Br₂N₂O₆Si⁻ [M-H]⁻ m/z 751.0480 [M-H]⁻, foundm/z 753.6495.

Synthesis of 7b-E and 7b-Z

Anhydrous solution of compound 6b (6.11 g, 8.1 mmol) in DMF (35 mL) wasadded dimethylphosphite (2.97 mL, 34.0 mmol) and triethylamine (2.26 mL,17.0 mmol) at 0° C., and then stirred over night at rt. After thesolution was diluted with ethyl acetate, the organic solution was washedwith sat. NH₄Cl aq. and brine. Then the organic solution was dried overMgSO₄, filtered and evaporated, and obtained crude material was purifiedrepeatedly by silica gel column chromatography (hexane/ethyl acetate,9:1 to 1:1) until all pure isomeric compound were collected separately,giving compound 7b-E (3.0 g, 55%); ¹H NMR (500 MHz, CDCl₃) δ 7.89-7.87(m, 2H), 7.70-7.62 (m, 5H), 7.51-7.39 (m, 8H), 7.10 (d, 1H, J_(HH)=8.3Hz), 6.47 (dd, 1H, J_(HH)=13.6, 0.8 Hz), 6.01 (dd, 1H, J_(HH)=13.6, 7.9Hz), 5.76-5.74 (m, 2H), 4.51 (dd, 1H, J_(HH)=7.8, 7.8 Hz), 7.36 (dd, 1H,J_(HH)=7.8 Hz, 4.9 Hz), 3.34 (s, 3H), 3.17 (dd, 1H, J_(HH)=4.7, 1.2 Hz),1.09 (s, 9H); ¹³C NMR (126 Hz, CDCl₃) δ 168.3, 161.7, 148.7, 138.4,135.9, 135.8, 135.3, 133.8, 132.6, 132.4, 131.2, 130.5, 130.4, 130.3,129.2, 128.0, 127.9, 112.1, 102.3, 88.9, 82.8, 82.6, 77.2, 74.2, 58.1,26.8, 19.1; and 7b-Z (1.23 g, 22%); ¹H NMR (500 MHz, CDCl₃) δ 7.89-7.87(m, 2H), 7.72-7.70 (m, 2H), 7.68-7.63 (m, 3H), 7.51-7.44 (m, 4H),7.41-7.37 (m, 4H), 7.16 (d, 1H, J=8.2 Hz), 6.53 (dd, 1H, J_(HH)=7.4, 0.6Hz), 6.03 (dd, 1H, J_(HH)=8.5, 7.4 Hz), 5.75-5.73 (m, 2H), 5.12 (t, 1H,J_(HH)=8.1 Hz), 3.93 (dd, 1H, J_(HH)=6.9, 5.0 Hz), 3.32 (br, 1H), 3.26(s, 3H), 1.10 (s, 9H); ¹³C NMR (126 Hz, CDCl₃) δ 168.3, 161.8, 148.7,139.3, 135.91, 135.85, 135.22, 132.74, 132.71, 131.2, 130.8, 130.5,130.23, 130.16, 129.2, 127.78, 127.75, 114.6, 102.2, 90.1, 82.4, 80.6,77.2, 74.8, 58.1, 26.8, 19.2.

Synthesis of Compound 8b

Anhydrous 5′-O-DMTr-2′-deoxy-2′-fluoro-3′-[methyl-N,N-(diisopropyl)amino]-phosphoramidite (4.26 g, 6.0 mmol) was dissolved in 0.45 M1H-tetrazole/CH₃CN solution (27 mL, 12 mmol) and stirred for 30 min atrt. To this solution, H₂O (3.6 mL) was added and stirred for 30 min atrt. After diluting with ethyl acetate, the organic solution was washedwith brine six times, dried over MgSO₄, filtered and then evaporated.Obtained compound 8b with a slight amount of impurity was used for thenext reaction without further purification; ³¹P NMR (CDCl₃, 202 MHz) δ8.92, 8.28.

Synthesis of Compound 9b

Anhydrous compound 7b-E (2.84 g, 4.20 mmol) and Pd(OAc)₂ (188.6 mg, 0.84mmol) and [1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium (II)(931.4 mg, 1.68 mmol) were purged with argon, and then dissolved intoanhydrous THF (50 mL). After adding propylene oxide (2.94 mL. 42.0mmol), compound 9b (3.16 g, 5.04 mmol) was added in one portion andstirred at for 4 h at 70° C. After removing solvent under reducedpressure, the crude mixture was purified by silica gel columnchromatography (hexane-ethyl acetate, 50:50 to 0:100) and obtainedfractions containing compound 9b were further purified by silica gelcolumn chromatography (1% TEA-CH₂Cl₂/MeOH, 0% to 5%) yielding compound9b as a mixture of diastereoisomers (3.3 g, 64%); ³¹P NMR (202 MHz,CDCl₃) δ 19.31, 18.72.

Synthesis of Compound 10b

Compound 9b (3.3 g, 2.70 mmol) in anhydrous THF (36.5 mL) was added 1.0M TBAF-THF (4.05 mL, 4.05 mmol) and stirred at ambient temperature for30 min. After diluting with CH₂Cl₂ (150 mL), the organic layer waswashed with brine, dried over MgSO₄, filtered, and then evaporated.Obtained crude material was purified by silica gel column chromatography(1% TEA-CH₂Cl₂/MeOH, 0% to 8%) yielding compound 10b (1.25 g, 47%); ³¹PNMR (202 MHz, CDCl₃) δ 19.8, 19.1.

Synthesis of Compound 11b

Compound 10b (393.2 mg, 0.4 mmol) was rendered anhydrous by repeatedco-evaporation with anhydrous CH₃CN and then dissolved into anhydrousCH₂Cl₂ (4.0 mL). To this solution N,N-diisopropylethylamine (0.21 mL,1.2 mmol) and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (0.11mL, 0.48 mmol) were added at 0° C. After stirring for 30 min at 0° C.,the reaction mixture was diluted with excess CH₂Cl₂. The organic layerwas repeatedly washed with aq. sat. NaHCO₃, dried over MgSO₄, filtered,and evaporated. The obtained crude material was purified by silica gelcolumn chromatography (1% TEA-CH₂C2/MeOH, from 100% to 4%) yieldingcompound 11b as a white foam (319.6 mg, 68%); ³¹P NMR (202 MHz, CDCl₃) δ150.7, 150.4, 150.3, 19.9, 19.5, 19.4, 18.8.

Example 5. Synthesis of Monomers for Ex-NA Intersubunit Linkage

As described supra, protection, deprotection, oxidation, olefination,and hydroboration oxidation is employed to achieve the free alcohol withan extended carbon chain shown in FIG. 6. Protection of the primaryalcohol followed by selective deprotection of the secondary alcoholallows for selective functionalization with a phosphanamine.

According to FIG. 6., 5′-O-DMTr protected starting material will befirst protected by TBDMS, then followed by 5′-O-detritylation. Obtainedcompound will be next oxidized to aldehyde by using IBX, then applied toWittig olefination using methyltriphenylphosphonium bromide andtert-BuOK in anhydrous THF solution to yield vinyl substitutednucleoside derivatives. This vinyl group will be reacted with 9-BBN tohave boronated intermediate then forwarded to oxidation by sodiumperborate yielding exNA structure with 6′-hydroxyl group. This hydroxylgroup will be first protected by DMTr group, and without silica gelcolumn purification, followed by deprotection of 3′-O-TBDMS group by 0.1M TBAF-THF solution. Obtained 6′-O-DMTr nucleoside derivatives will bephosphitylated to yield methyl protected phosphoramidites. Each stepwill be first quenched and extracted followed by purification by silicagel column chromatography except for the first 3′-O-TBDMS protectionstep.

FIG. 7 provides a synthetic procedure for achieving another monomer forex-NA-modified intersubunit linkages. As discussed above, Jonesoxidation, Wittig olefination, hydroboration oxidation, followed bymesylation and Finkelstein iodination is employed to achieve advancedintermediate monomers. Nucleophilic substitution with a phosphane andsubsequent methylation produces the monomer shown in FIG. 7.

Example 6. Vinyl Phosphonate Walk on Guide Strand

The sequences having VP-modifications generated herein are shown belowand in FIG. 14. Antisense strands are depicted 5′ to 3′, with the SNPsite in bold and the mismatch in italics.

^(VP)Guide strands Name Sequence (5′ -> 3′) ^(VP)G1 HTT10150_1/2P[mU-fU]#(mA)(fA)(mU)(fC)(mU)(fC)(mU)(fU)(mU)(fA)(mC)#(fU)#(mG)#(fA)#(mU)#(fA)#(mU)#(fA) ^(VP)G2 HTT10125_2/3P(mU)#[fU-mU](fU)(mU)(fA)(mA)(fA)(mU)(fC)(mC)(fU)(mG)#(fA)#(mG)#(fA)#(mA)#(fG)#(mA)#(fA) ^(VP)G3 HTT10125_3/4P(mU)#(fU)#[mU-fU](mU)(fA)(mA)(fA)(mU)(fC)(mC)(fU)(mG)#(fA)#(mG)#(fA)#(mA)#(fG)#(mA)#(fA) ^(VP)G4 HTT10125_4/5P(mU)#(fU)#(mU)[fU-mU](fA)(mA)(fA)(mU)(fC)(mC)(fU)(mG)#(fA)#(mG)#(fA)#(mA)#(fG)#(mA)#(fA) ^(VP)G5 HTT10146_5/6P(mU)#(fC)#(mU)(fC)[mU-fU](mU)(fA)(mC)(fU)(mG)(fA)(mU)#(fA)#(mU)#(fA)#(mA)#(fU)#(mU)#(fA) ^(VP)G6 HTT462_6/7P(mU)#(fA)#(mU)(fG)(mU)[fU-mU](fU)(mC)(fA)(mC)(fA)(mU)#(fA)#(mU)#(fU)#(mG)#(fU)#(mC)#(fA) ^(VP)G7 HTT8603_7/8P(mU)#(fG)#(mA)(fA)(mU)(fG)[mU-fU](mC)(fA)(mC)(fG)(mC)#(fA)#(mG)#(fU)#(mG)#(fG)#(mG)#(fC) ^(VP)G8 HTT_exon_1_150_8/9P(mU)#(fA)#(mU)(fC)(mA)(fG)(mC)[fU-mU](fU)(mU)(fC)(mC)#(fA)#(mG)#(fG)#(mG)#(fU)#(mC)#(fG) ^(VP)G9 HTT10150_9/10P(mU)#(fU)#(mA)(fA)(mU)(fC)(mU)(fC)[mU-fU](mU)(fA)(mC)#(fU)#(mG)#(fA)#(mU)#(fA)#(mU)#(fA) ^(VP)G10 HTT10150_10/11P(mU)#(fU)#(mA)(fA)(mU)(fC)(mU)(fC)(mU)[fU-mU](fA)(mC)#(fU)#(mG)#(fA)#(mU)#(fA)#(mU)#(fA) ^(VP)G11 HTT1219_11/12P(mU)#(fU)#(mA)(fA)(mC)(fG)(mU)(fC)(mA)(fG)[mU-fU](mC)#(fA)#(mU)#(fA)#(mA)#(fA)#(mC)#(fC) ^(VP)G12 HTT467_12/13P(mU)#(fC)#(mC)(fA)(mC)(fU)(mA)(fU)(mG)(fU)(mU)[fU-mU]#(fC)#(mA)#(fC)#(mA)#(fU)#(mA)#(fU) ^(VP)G13 HTT1907_13/14P(mU)#(fC)#(mC)(fA)(mA)(fA)(mU)(fA)(mC)(fU)(mG)(fG)[mU-fU]#(mG)#(fU)#(mC)#(fG)#(mG)#(fU) ^(VP)G14 HTT1257_14/15P(mU)#(fC)#(mC)(fG)(mG)(fU)(mC)(fA)(mC)(fA)(mA)(fC)(mA)#[fU-mU]#(fG)#(mU)#(fG)#(mG)#(fU) ^(VP)G15 HTT462_15/16P(mU)#(fA)#(mU)(fG)(mU)(fU)(mU)(fU)(mC)(fA)(mC)(fA)(mU)#(fA)#[mU-fU]#(mG)#(fU)#(mC)#(fA) ^(VP)G16 HTT460_16/17P(mU)#(fU)#(mU)(fG)(mG)(fU)(mA)(fG)(mC)(fU)(mG)(fA)(mA)#(fA)#(mG)#[fU-mU]#(fC)#(mU)#(fU) ^(VP)G17 HTT10150-mod_17/18P(mU)#(fU)#(mA)(fA)(mU)(fC)(mU)(fC)(mU)(fU)(mU)(fA)(mC)#(fU)#(mG)#(fA)#[mU-fU]#(mU)#(fA) ^(VP)G18 HTT10146_18/19P(mU)#(fC)#(mU)(fC)(mU)(fU)(mU)(fA)(mC)(fU)(mG)(fA)(mU)#(fA)#(mU)#(fA)#(mA)#[fU-mU]#(fA) ^(VP)G19 HTT10150-P(mU)#(fU)#(mA)(fA)(mU)(fC)(mU)(fC)(mU)(fU)(mU)(fA)(mC)#(fU)#(mG)#mod_19/20 (fA)#(mU)#(fA)#[mU-fU] ^(VP)G20 HTT10150-P[mU-fU]#(mA)(fA)(mU)(fC)(mU)(fC)(mU)(fU)(mU)(fA)(mC)#(fU)#(mG)#(fA)#mod_1/2_17/18_19/20 [mU-fU]#[mU-fU] P: phosphate; (mU, G, A or C):2′-OMe-RNA; (fU, G, A or C): 2′-Fluoro-2′-deoxy-RNA; [mU-fU]: mU-fUdimer linked with vinylphosphonate; [fU-mU]: fU-MU dimer linked withvinylphosphonate; #: phosphorothiate linkage

Example 7. Thermal Stability Assays

The results of the thermal stability assays for modifiedoligonucleotides and duplex RNA are provided in FIGS. 9A and 9B. Theoligonucleotides and RNA tested contained vinyl phosphonatemodifications in the intersubunit linkages at varying positions.

1 μM guide strand and 1 μM complementary sense strand were annealed in a10 mM sodium phosphate buffer (pH 7) containing 100 mM NaCl by heatingat 95° C. for 1 min and cooled down gradually to room temperature. Tmmeasurement was performed with temperature controller. Both the heatingand cooling curves were measured over a temperature range from to 95° C.at 1.0° C./min for three times. The absorbance at 260 nm was recorded atevery temperature point.

Example 8. Digestive Stability Assays

The results of the digestive stability assays for modified RNA areprovided in FIGS. 10A-10C. The RNA tested contained VP and PSmodifications in the intersubunit linkages. RNA having a VP-modificationshowed higher stability against 5′- and 3′-exonucleases. In the case ofXRN1, RNA having VP-modified linkers were better substrates than RNAhaving natural phosphate linkers.

XRN-1 Stability Measurement

2.5 uM RNA single strands (50 pmol) were incubated in RNase-free wateror with 0.17 U or 3.3 U of Terminator™ (EpiCentre) exonuclease at 37° C.in buffer A (EpiCentre, provided with Terminator™ enzyme). Part of thereaction mixture (4 pmol) was taken at each time point (3, 5, 10, 15,30, 60, 120 min) and quenched by adding 95% formamide containing 20 mMEDTA, subsequently frozen by liquid nitrogen, and then kept at −80° C.Right before applying the collected samples to denatured gel, sampleswere heated at 95° C. for 1 min, iced, and then applied to the 20%formamide 20% polyacrylamide gel containing 7 M urea. After gelelectrophoresis at 500 V for 3 h, gels were stained with SYBR® GoldNucleic Acid Gel Stain (Thermo Fisher Scientific) and visualized byTyphoon FLA 9000 (GE Healthcare).

SVPD Stability Measurement

17.5 uM RNA single strands (50 pmol) were incubated in RNase-free wateror in a 10 mM Tris-HCl buffer (pH 8.0) containing 2.0 mM MgCl₂ and 4mU/mL (or 10 mU/mL) of snake venom phosphodiesterase I (SVPD,Sigma-Aldrich). Part of the reaction mixture (4 pmol) was taken at eachtime point (5 min, 10 min, 30 min, 1 h, 2 h, 4 h, 8 h) and quenched byadding 95% formamide containing 20 mM EDTA, subsequently frozen byliquid nitrogen, and then kept at −80° C. Right before applying thecollected samples to denatured gel, samples were heated at 95° C. for 1min, iced, and then applied to the 20% formamide 20% polyacrylamide gelcontaining 7 M urea. After gel electrophoresis at 500 V for 2 h, gelswere stained with SYBR® Gold Nucleic Acid Gel Stain (Thermo FisherScientific) and visualized by Typhoon FLA 9000 (GE Healthcare).

BSP Stability Measurement

10 uM RNA was incubated at 37° C. in RNase-free water or 30 mM NaOAc (pH6.0) buffer containing 0.25 U/mL Phosphodiesterase II from bovine spleen(BSP). Part of the reaction mixture (4 pmol) was taken at each timepoint (5 min, 10 min, 30 min, 1 h, 2 h, 4 h, 8 h) and quenched by adding95% formamide containing 20 mM EDTA, subsequently frozen by liquidnitrogen, and then kept at −80° C. Right before applying the collectedsamples to denatured gel, samples were heated at 95° C. for 1 min, iced,and then applied to the 20% formamide 20% polyacrylamide gel containing7 M urea. After gel electrophoresis at 120 V for 12 hours, gels werestained with SYBR® Gold Nucleic Acid Gel Stain (Thermo FisherScientific) and visualized by Typhoon (GE Healthcare).

Example 9. Allelic Discrimination

The results of allelic discrimination assays are summarized in FIG. 11.As can be seen in FIG. 11, adding a mismatch in the siRNA sequenceimproves allelic discrimination without impairing mutant allelesilencing. In other words, the combination of a VP-modification and anadditional mismatch can completely abolish off-target silencing withoutlosing efficacy of on-target silencing.

Example 10. Silencing Efficacy Assays

FIG. 12 exemplifies the effect a vinyl phosphonate modification in anintersubunit linkage at varying positions on the guide strand has onsilencing. The data in FIG. 12 indicate that RISC is very sensitive toVP modifications and therefore the combination of having a VPmodification as well as mismatched base pairing can be a useful strategyto reduce off-target effects. VP insertion of the siRNA guide strandshowed significant position-dependent impacts on siRNA potency.

FIGS. 16 and 17 also demonstrate VP-modified siRNAs' ability to silenceand knockdown mRNA. Passive uptake of siRNAs was conducted (7 pointdose) with a three day incubation period. The remaining target mRNA wasquantified using Quantigene™ 2.0 RNA.

A method for measuring the data in FIG. 12 is described below.

hsiRNA Passive Delivery.

Cells were plated in Dulbecco's Modified Eagle's Medium containing 6%FBS at 8,000 cells per well in 96-well cell culture plates. hsiRNAs werediluted to twice the final concentration in OptiMEM (Carlsbad, Calif.;31985-088), and 50 μL diluted hsiRNAs were added to 50 μL of cells,resulting in 3% FBS final. Cells were incubated for 72 hours at 37° C.and 5% CO₂. The maximal dose in the in vitro dose response assays was1.5 M compound,

Method for Quantitative Analysis of Target mRNA.

mRNA was quantified from cells using the QuantiGene 2.0 assay kit(Affymetrix, QS0011). Cells were lysed in 250 μL diluted lysis mixturecomposed of one part lysis mixture (Affymetrix, 13228), two parts H₂Oand 0.167 μg/μL proteinase K (Affymetrix, QS0103) for 30 min at 55° C.Cell lysates were mixed thoroughly, and 40 μL of each lysate was addedper well of a capture plate with 20 μL diluted lysis mixture withoutproteinase K. Probe sets for human HTT and HPRT (Affymetrix; #SA-50339,SA-10030) were diluted and used according to the manufacturer'srecommended protocol. Datasets were normalized to HPRT.

Method for Creating Bar Graph

Data were analyzed using GraphPad Prism 7 software (GraphPad Software,Inc., San Diego, Calif.). Concentration-dependent IC₅₀ curves werefitted using a log(inhibitor) versus response-variable slope (fourparameters). For each cell treatment plate, the level of knockdown ateach dose was normalized to the mean of the control group (untreatedgroup). The lower limit of the curve was set to less than 5, and theupper limit of the curve was set to greater than 95. To create the bargraph, the percent difference was calculated by subtracting the IC₅₀value for each compound from the IC₅₀ value for each correspondingcontrol compound, dividing by the IC₅₀ value for the control compound,and multiplying by 100. If the percent difference was less than −500%,the percent difference was artificially set to −500%. The lower limit ofthe graph was cut at −300%.

Example 11. Solid Phase Synthesis of RNA Oligonucleotide Modified withVinyl Phosphonate Linkage

A method of preparing RNA having the vinyl phosphonate modification inthe intersubunit linkage can be found in FIG. 13. FIG. 15 shows examplesof hsiRNA antisense scaffolds aligned to the HTT sequence surroundingSNP site rs362273 that have been synthesized. As such, a syntheticprotocol for internal VP-modified RNA has been established.

Synthesis of Inter-Nucleotide (E)-Vinyl Phosphonate Modified RNAOligonucleotides

The synthesis of RNA oligonucleotides having one vinyl phosphonatelinkage was performed on MerMade 12 automated RNA synthesizer(BioAutomation) using 0.1 M anhydrous CH₃CN solution of 2′-modified(2′-fluoro, 2′-O-methyl) phosphoramidites and vinylphosphonate-linkeddimer phosphoramidites. For the solid support, UnyLinker support(ChemGenes) was used. The synthesis was conducted by standard 1.0 μmolscale RNA phosphoramidite synthesis cycle, which consists of (i)detritylation, (ii) coupling, (iii) capping, and (iv) iodine oxidation.5-(Benzylthio)-1H-tetrazole in anhydrous CH₃CN was used forphosphoramidite activating reagent, and 3% dichloroacetic acid in CH₂Cl₂was used for detritylation. 16% N-methylimidazole in tetrehydrofurane(Cap A) and 80:10:10 (v/v/v) tetrhydrofurane-Ac₂O-2,6-lutidine (Cap B)were used for capping reaction. 0.02 M 12 in THF-pyridine-H₂O (7:2:1,v/v/v) was used for oxidation and 0.1 M3-[(Dimethylamino-methylidene)amino]-3H-1,2,4-dithiazole3-thione inpyridine:CH₃CN (9:1, v/v) was used for sulfurizing. For 5′-terminalphosphorylation, bis(2-cyanoethyl)-N,N-diisopropyl phosphoramidite wasused. For the 3′-cholesterol modified RNA oligonucleotide synthesis,cholesterol 3′-lcaa CPG 500 Å (ChemGenes) was used, and RNA synthesiswas conducted in the same condition as the condition used forVP-modified RNAs. After the chemical chain elongation, deprotection andcleavage from the solid support were conducted by NH₄OH-EtOH (3:1, v/v)for 48 h at 26° C. In the case of vinyl phosphonate modified RNA, RNA onsolid support was first treated with TMSBr-pyridine-CH₂Cl₂ (3:1:18,v/v/v) for 1 h at ambient temperature in RNA synthesis column. Solidsupport was then washed by water (1 mL×3), CH₃CN (1 mL×3) and CH₂Cl₂ (1mL×3) by flowing solution thorough synthesis column, and then driedunder vacuum. After transferring the solid support to screw-cappedsample tube, base treatment by NH₄OH-EtOH (3:1, v/v) for 48 h at 26° C.was conducted. Crude RNA oligonucleotide without cholesterol conjugatewas purified by standard anion exchange HPLC, whereas RNAs withcholesterol-conjugate were purified by reversed-phase HPLC. All purifiedRNAs were desalted by Sephadex G-25 (GE Healthcare) and characterized byelectrospray ionization mass spectrometry (ESI-MS) analysis.

Example 12. Oligonucleotides with Ex-NA Intersubunit Linkages

The ex-NA intersubunit linkages described above in Example 5 were usedin an oligonucleotide walk experiment, where each intersubunit linkagein an antisense and sense strand was modified with the ex-NAintersubunit linkage. Tables 4-10 below show the antisense and sensestrands used in this Example, as well as duplexes formed by differentcombinations of said antisense and sense strands. A novel synthesisscheme for generating ex-NA containing oligonucleotides was alsoemployed and shown in FIG. 65, FIG. 66, and FIG. 67.

TABLE 4 Antisense strands having ex-NA intersubunit linkages NameSequence (5′ -> 3′)^(a) ex-1 5′-P(ex_mU)#(fU)#(mA)(fA)(mU)(fC)(mU)(fC)(mU)(fU)(mU)(fA)(mC)#(fU)#(mG)#(fA)#(mU)#(fA)#(mU)#(fA) ex-2 5′-P(mU)#(ex_fU)#(mA)(fA)(mU)(fC)(mU)(fC)(mU)(fU)(mU)(fA)(mC)#(fU)#(mG)#(fA)#(mU)#(fA)#(mU)#(fA) ex-3 5′-P(mU)#(fU)#(ex_mU)(fU)(mU)(fA)(mA)(fA)(mU)(fC)(mC)(fU)(mG)#(fA)#(mG)#(fA)#(mA)#(fG)#(mA)#(fA) ex-4 5′-P(mU)#(fU)#(mU)(ex_fU)(mU)(fA)(mA)(fA)(mU)(fC)(mC)(fU)(mG)#(fA)#(mG)#(fA)#(mA)#(fG)#(mA)#(fA) ex-5 5′-P(mU)#(fU)#(mA)(fA)(ex_mU)(fC)(mU)(fC)(mU)(fU)(mU)(fA)(mC)#(fU)#(mG)#(fA)#(mU)#(fA)#(mU)#(fA) ex-6 5′-P(mU)#(fC)#(mC)(fA)(mC)(ex_fU)(mA)(fU)(mG)(fU)(mU)(fU)(mU)#(fC)#(mA)#(fC)#(mA)#(fU)#(mA)#(fU) ex-7 5′-P(mU)#(fU)#(mA)(fA)(mU)(fC)(ex_mU)(fC)(mU)(fU)(mU)(fA)(mC)#(fU)#(mG)#(fA)#(mU)#(fA)#(mU)#(fA) ex-8 5′-P(mU)#(fC)#(mC)(fA)(mC)(fU)(mA)(ex_fU)(mG)(fU)(mU)(fU)(mU)#(fC)#(mA)#(fC)#(mA)#(fU)#(mA)#(fU) ex-9 5′-P(mU)#(fU)#(mA)(fA)(mU)(fC)(mU)(fC)(ex_mU)(fU)(mU)(fA)(mC)#(fU)#(mG)#(fA)#(mU)#(fA)#(mU)#(fA) ex-10 5′-P(mU)#(fU)#(mA)(fA)(mU)(fC)(mU)(fC)(mU)(ex_fU)(mU)(fA)(mC)#(fU)#(mG)#(fA)#(mU)#(fA)#(mU)#(fA) ex-11 5′-P(mU)#(fU)#(mA)(fA)(mU)(fC)(mU)(fC)(mU)(fU)(ex_mU)(fA)(mC)#(fU)#(mG)#(fA)#(mU)#(fA)#(mU)#(fA) ex-12 5′-P(mU)#(fC)#(mC)(fA)(mC)(fU)(mA)(fU)(mG)(fU)(mU)(ex_fU)(mU)#(fC)#(mA)#(fC)#(mA)#(fU)#(mA)#(fU) ex-13 5′-P(mU)#(fC)#(mC)(fA)(mC)(fU)(mA)(fU)(mG)(fU)(mU)(fU)(ex_mU)#(fC)#(mA)#(fC)#(mA)#(fU)#(mA)#(fU) ex-14 5′-P(mU)#(fU)#(mA)(fA)(mU)(fC)(mU)(fC)(mU)(fU)(mU)(fA)(mC)#(ex_fU)#(mG)#(fA)#(mU)#(fA)#(mU)#(fA) ex-15 5′-P(mU)#(fG)#(mC)(fC)(mU)(fA)(mA)(fG)(mA)(fG)(mC)(fA)(mC)#(fA)#(ex_mU)#(fU)#(mU)#(fA)#(mG)#(fU) ex-16 5′-P(mU)#(fG)#(mC)(fC)(mU)(fA)(mA)(fG)(mA)(fG)(mC)(fA)(mC)#(fA)#(mU)#(ex_fU)#(mU)#(fA)#(mG)#(fU) ex-17 5′-P(mU)#(fU)#(mA)(fA)(mU)(fC)(mU)(fC)(mU)(fU)(mU)(fA)(mC)#(fU)#(mG)#(fA)#(ex_mU)#(fA)#(mU)#(fA) ex-18 5′-P(mU)#(fC)#(mC)(fA)(mC)(fU)(mA)(fU)(mG)(fU)(mU)(fU)(mU)#(fC)#(mA)#(fC)#(mA)#(ex_fU)#(mA)#(fU) ex-19 5′-P(mU)#(fU)#(mA)(fA)(mU)(fC)(mU)(fC)(mU)(fU)(mU)(fA)(mC)#(fU)#(mG)#(fA)#(mU)#(fA)#(ex_mU)#(fA) ex-20 5′-P(mU)#(fC)#(mC)(fA)(mC)(fU)(mA)(fU)(mG)(fU)(mU)(fU)(mU)#(fC)#(mA)#(fC)#(mA)#(fU)#(mA)#(ex_fU) ^(a)(mN): 2′-OMe, (fN): 2′-Fluoro, (ex_mU):2′-OMe-ex-uridine, (ex_fU): 2′-fluoro-ex-uridine, P: Phosphate, #:Phosphorothioate

TABLE 5 Sense strands having ex-NA intersubunit linkages Name Sequence(5′ -> 3′)^(a) ex-SS-15′-(ex_fU)#(mG)#(fA)(mA)(fA)(mA)(fC)(mA)(fU)(mA)(fG)(mU)(fG)#(mG)#(fA)-TegChol ex-SS-25′-(fC)#(ex_mU)#(fC)(mA)(fG)(mG)(fA)(mU)(fU)(mU)(fA)(mA)(fA)#(mA)#(fA)-TegChol ex-SS-35′-(fA)#(mA)#(ex_fU)(mG)(fU)(mU)(fG)(mU)(fG)(mA)(fC)(mC)(fG)#(mG)#(fA)-TegChol ex-SS-45′-(fC)#(mA)#(fG)(ex_mU)(fA)(mA)(fA)(mG)(fA)(mG)(fA)(mU)(fU)#(mA)#(fA)-TegChol ex-SS-55′-(fA)#(mA)#(fU)(mG)(ex_fU)(mU)(fG)(mU)(fG)(mA)(fC)(mC)(fG)#(mG)#(fA)-TegChol ex-SS-65′-(fA)#(mA)#(fU)(mG)(fU)(ex_mU)(fG)(mU)(fG)(mA)(fC)(mC)(fG)#(mG)#(fA)-TegChol ex-SS-75′-(fA)#(mU)#(fG)(mU)(fG)(mC)(ex_fU)(mC)(fU)(mU)(fA)(mG)(fG)#(mC)#(fA)-TegChol ex-SS-85′-(fC)#(mU)#(fC)(mA)(fG)(mG)(fA)(ex_mU)(fU)(mU)(fA)(mA)(fA)#(mA)#(fA)-TegChol ex-SS-95′-(fC)#(mU)#(fC)(mA)(fG)(mG)(fA)(mU)(ex_fU)(mU)(fA)(mA)(fA)#(mA)#(fA)-TegChol ex-SS-105′-(fC)#(mU)#(fC)(mA)(fG)(mG)(fA)(mU)(fU)(ex_mU)(fA)(mA)(fA)#(mA)#(fA)-TegChol ex-SS-115′-(fC)#(mU)#(fG)(mG)(fA)(mA)(fA)(mA)(fG)(mC)(ex_fU)(mG)(fA)#(mU)#(fA)-TegChol ex-SS-125′-(fC)#(mA)#(fG)(mU)(fA)(mA)(fA)(mG)(fA)(mG)(fA)(ex_mU)(fU)#(mA)#(fA)-TegChol ex-SS-135′-(fC)#(mA)#(fG)(mU)(fA)(mA)(fA)(mG)(fA)(mG)(fA)(mU)(ex_fU)#(mA)#(fA)-TegChol ex-SS-145′-(fC)#(mU)#(fG)(mG)(fA)(mA)(fA)(mA)(fG)(mC)(fU)(mG)(fA)#(ex_mU)#(fA)-TegChol ex-SS-155′-(fC)#(mA)#(fG)(mU)(fA)(mA)(fA)(mG)(fA)(mG)(fA)(mU)(fU)#(mA)#(ex_fU)-TegChol ^(a)(mN): 2′-OMe, (fN): 2′-Fluoro, (ex_mU): 2′-OMe-ex-uridine,(ex_fU): 2′-fluoro-ex-uridine, P: Phosphate, #: Phosphorothioate,TegChol: Tetraethyleneglycol-linked cholesterol

TABLE 6 Control antisense strands Name Sequence (5′ -> 3′)^(a) AS-05′-P(mU)(fU)(mA)(fA)(mU)(fC)(mU)(fC)(mU)(fU)(mU)(fA)(mC)(fU)(mG)(fA)(mU)(fU)(mU)(fU) AS-1 5′-P(mU)#(fU)#(mA)(fA)(mU)(fC)(mU)(fC)(mU)(fU)(mU)(fA)(mC)#(fU)#(mG)#(fA)#(mU)#(fA)#(mU)#(fA) AS-2 5′-P(mU)#(fU)#(mA)(fA)(mU)(fC)(mU)(fC)(mU)(fU)(mU)(fA)(mC)#(fU)#(mG)#(fA)#(mU)#(fU)#(mU)#(fU) AS-3 5′-P(mU)#(fU)#(mU)(fU)(mU)(fA)(mA)(fA)(mU)(fC)(mC)(fU)(mG)#(fA)#(mG)#(fA)#(mA)#(fG)#(mA)#(fA) AS-4 5′-P(mU)#(fC)#(mC)(fA)(mC)(fU)(mA)(fU)(mG)(fU)(mU)(fU)(mU)#(fC)#(mA)#(fC)#(mA)#(fU)#(mA)#(fU) AS-5 5′-P(mU)#(fG)#(mC)(fC)(mU)(fA)(mA)(fG)(mA)(fG)(mC)(fA)(mC)#(fA)#(mU)#(fU)#(mU)#(fA)#(mG)#(fU) AS-6 5′-P(mU)#(fA)#(mU)(fC)(mA)(fG)(mC)(fU)(mU)(fU)(mU)(fC)(mC)#(fA)#(mG)#(fG)#(mG)#(fU)#(mC)#(fG) AS-7 5′-P(mU)#(fC)#(mC)(fG)(mG)(fU)(mC)(fA)(mC)(fA)(mA)(fC)(mA)#(fU)#(mU)#(fG)#(mU)#(fG)#(mG)#(fU) AS-8 5′-P(mA)#(fU)#(mA)(fA)(mU)(fC)(mU)(fC)(mU)(fU)(mU)(fA)(mC)#(fU)#(mG)#(fA)#(mU)#(fA)#(mU)#(fA) ^(a)(mN): 2′ -OMe, (fN): 2′-Fluoro, P: Phosphate, #:Phosphorothioate

TABLE 7 Control sense strands Name Sequence (5′ -> 3′)^(a) SS-15′-(fC)#(mA)#(fG)(mU)(fA)(mA)(fA)(mG)(fA)(mG)(fA)(mU)(fU)#(mA)#(fA)-TegCholSS-25′-(fC)#(mU)#(fC)(mA)(fG)(mG)(fA)(mU)(fU)(mU)(fA)(mA)(fA)#(mA)#(fA)-TegCholSS-35′-(fU)#(mG)#(fA)(mA)(fA)(mA)(fC)(mA)(fU)(mA)(fG)(mU)(fG)#(mG)#(fA)-TegCholSS-45′-(fA)#(mU)#(fG)(mU)(fG)(mC)(fU)(mC)(fU)(mU)(fA)(mG)(fG)#(mC)#(fA)-TegCholSS-55′-(fC)#(mU)#(fG)(mG)(fA)(mA)(fA)(mA)(fG)(mC)(fU)(mG)(fA)#(mU)#(fA)-TegCholSS-65′-(fA)#(mA)#(fU)(mG)(fU)(mU)(fG)(mU)(fG)(mA)(fC)(mC)(fG)#(mG)#(fA)-TegCholSS-75′-(fC)#(mA)#(fG)(mU)(fA)(mA)(fA)(mG)(fA)(mG)(fA)(mU)(fU)#(mA)#(fU)-TegChol^(a)(m): 2′-OMe, (fN): 2′-Fluoro, #: Phosphorothioate, TegChol:Tetraethyleneglycol-linked cholesterol

TABLE 8 siRNA duplexes (D1-D20) having ex-NA modified antisense strandsGroup 1 exNA modified Corresponding exNA walk on Duplex Antisense Sensecontrol duplex# Antisense strand # strand strand (See Group 3) D1 ex-1SS-1 D40 D2 ex-2 SS-1 D40 D3 ex-3 SS-2 D42 D4 ex-4 SS-2 D42 D5 ex-5 SS-1D40 D6 ex-6 SS-3 D43 D7 ex-7 SS-1 D40 D8 ex-8 SS-3 D43 D9 ex-9 SS-1 D40D10 ex-10 SS-1 D40 D11 ex-11 SS-1 D40 D12 ex-12 SS-3 D43 D13 ex-13 SS-3D43 D14 ex-14 SS-1 D40 D15 ex-15 SS-4 D44 D16 ex-16 SS-4 D44 D17 ex-17SS-1 D40 D18 ex-18 SS-3 D43 D19 ex-19 SS-1 D40 D20 ex-20 SS-3 D43

TABLE 9 siRNA duplexes (D25-D39) having ex-NA modified sense strandsGroup 2 exNA Corresponding exNA walk on Duplex Antisense modifiedcontrol duplex# Sense strand # strand Sense strand (See Group 3) D25AS-3 ex-SS-1 D43 D26 AS-2 ex-SS-2 D42 D27 AS-6 ex-SS-3 D46 D28 AS-1ex-SS-4 D40 D29 AS-6 ex-SS-5 D46 D30 AS-6 ex-SS-6 D46 D31 AS-4 ex-SS-7D44 D32 AS-2 ex-SS-8 D42 D33 AS-2 ex-SS-9 D42 D34 AS-2 ex-SS-10 D42 D35AS-5 ex-SS-11 D45 D36 AS-1 ex-SS-12 D40 D37 AS-1 ex-SS-13 D40 D38 AS-5ex-SS-14 D45 D39 AS-7 ex-SS-15 D47

TABLE 10 Control siRNA duplexes Group3 Control Duplex Antisense SenseCorresponding duplexes # strands strands exNA-duplexes D40 AS-1 SS-1 D1,2, 5, 7, 9, 10, 11, 14, 17, 19, D28, 36, 37 D42 AS-3 SS-2 D3, 4, D26,32, 33, 34 D43 AS-4 SS-3 D6, 8, 12, 13, 18, 20, D25 D44 AS-5 SS-4 D15,16, D31 D45 AS-6 SS-5 D35, 38 D46 AS-7 SS-6 D27, 29, 30 D47 AS-8 SS-7D39

The siRNA duplexes recited above were used in in vitro mRNA silencingexperiments to determine relative silencing efficacy. Experimentaldetails are described below.

In Vitro Screen.

1.5 μM siRNAs were passively delivered to cells. Cells were plated inDulbecco's Modified Eagle's Medium containing 6% FBS at 8,000 cells perwell in 96-well cell culture plates. siRNAs were diluted to twice thefinal concentration in OptiMEM (Carlsbad, Calif.; 31985-088), and 50 μLdiluted siRNAs were added to 50 μL of cells, resulting in 3% FBS final.Cells were incubated for 72 hours at 37° C. and 5% CO₂.

Quantitative Analysis of Target mRNA.

mRNA was quantified from cells using the QuantiGene 2.0 assay kit(Affymetrix, QS0011). Cells were lysed in 250 μL diluted lysis mixturecomposed of one part lysis mixture (Affymetrix, 13228), two parts H2Oand 0.167 μg/μL proteinase K (Affymetrix, QS0103) for 30 min at 55° C.Cell lysates were mixed thoroughly, and 40 μL of each lysate was addedper well of a capture plate with 40 μL diluted lysis mixture withoutproteinase K and 20 μL diluted probe set. Probe sets for human HTT andHypoxanthine Phosphoribosyltransferase (HPRT) (Affymetrix; #SA-50339,SA-10030) were diluted and used according to the manufacturer'srecommended protocol. Datasets were normalized to HPRT

Cell Treatment: Reporter Assay.

HeLa cells were grown and maintained in Gibco DMEM (ref.#11965-092) with1% pen/strep and 10% heat inactivated FBS. Three days prior totreatment, two 10 cm² dishes were plated with 2×10⁶ HeLa cells. Thefollowing day, DMEM was replaced with Gibco OptiMEM (ref. #31985-070)and 6 μg of reporter plasmid was added to cells using InvitrogenLipofectamine 3000 (ref. #L3000-015), following the manufacturer'sprotocol. Cells were left in OptiMEM/lipofectamine overnight to allowfor maximum reporter plasmid transfection. The following day, siRNA wasdiluted in Opti-MEM and added to 96-well white wall clear bottom tissueculture plate, in triplicate, for each reporter plasmid. HeLa cellstransfected with reporter plasmids the night prior were resuspended inDMEM with 6% heat inactivated FBS (no pen/strep) at 0.15×10⁶ cells/mLand added to plate containing siRNA.

Cells were lysed after 72 hours of treatment (100% confluency) with 1×Passive Lysis Buffer from Dual-Luciferase Assay System Pack (Promegaref. #E1960). Following lysis, luminescence was read after addition of50 μl Luciferase Assay Reagent II (Promega ref. #E1960), then read asecond time after addition of 50 μL/well of Stop and Glow reagent(Promega ref. #E1960). Absorbances were normalized to untreated controlsand graphed on a log scale.

As shown in FIG. 64, all tested siRNA duplexes effectively silenced thetarget HTT mRNA. Moreover, numerous siRNA duplexes silenced the targetmRNA as well as the control duplex siRNA. This data provides the firstexample of an ex-NA internucleotide linkage incorporated into anoligonucleotide strand.

INCORPORATION BY REFERENCE

The contents of all cited references (including literature references,patents, patent applications, and websites) that maybe cited throughoutthis application are hereby expressly incorporated by reference in theirentirety for any purpose, as are the references cited therein. Thedisclosure will employ, unless otherwise indicated, conventionaltechniques of immunology, molecular biology and cell biology, which arewell known in the art.

The present disclosure also incorporates by reference in their entiretytechniques well known in the field of molecular biology and drugdelivery. These techniques include, but are not limited to, techniquesdescribed in the following publications:

-   Atwell et al. J. Mol. Biol. 1997, 270: 26-35;-   Ausubel et al. (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John    Wiley &Sons, N Y (1993);-   Ausubel, F. M. et al. eds., SHORT PROTOCOLS IN MOLECULAR BIOLOGY    (4th Ed. 1999) John Wiley & Sons, NY. (ISBN 0-471-32938-X);-   CONTROLLED DRUG BIOAVAILABILITY, DRUG PRODUCT DESIGN AND    PERFORMANCE, Smolen and Ball (eds.), Wiley, New York (1984);-   Giege, R. and Ducruix, A. Barrett, CRYSTALLIZATION OF NUCLEIC ACIDS    AND PROTEINS, a Practical Approach, 2nd ea., pp. 20 1-16, Oxford    University Press, New York, N.Y., (1999);-   Goodson, in MEDICAL APPLICATIONS OF CONTROLLED RELEASE, vol. 2, pp.    115-138 (1984);-   Hammerling, et al., in: MONOCLONAL ANTIBODIES AND T-CELL HYBRIDOMAS    563-681 (Elsevier, N.Y., 1981;-   Harlow et al., ANTIBODIES: A LABORATORY MANUAL, (Cold Spring Harbor    Laboratory Press, 2nd ed. 1988);-   Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST    (National Institutes of Health, Bethesda, Md. (1987) and (1991);-   Kabat, E. A., et al. (1991) SEQUENCES OF PROTEINS OF IMMUNOLOGICAL    INTEREST, Fifth Edition, U.S. Department of Health and Human    Services, NIH Publication No. 91-3242;-   Kontermann and Dubel eds., ANTIBODY ENGINEERING (2001)    Springer-Verlag. New York. 790 pp. (ISBN 3-540-41354-5).-   Kriegler, Gene Transfer and Expression, A Laboratory Manual,    Stockton Press, N Y (1990);-   Lu and Weiner eds., CLONING AND EXPRESSION VECTORS FOR GENE FUNCTION    ANALYSIS (2001) BioTechniques Press. Westborough, Mass. 298 pp.    (ISBN 1-881299-21-X).-   MEDICAL APPLICATIONS OF CONTROLLED RELEASE, Langer and Wise (eds.),    CRC Pres., Boca Raton, Fla. (1974);-   Old, R. W. & S. B. Primrose, PRINCIPLES OF GENE MANIPULATION: AN    INTRODUCTION TO GENETIC ENGINEERING (3d Ed. 1985) Blackwell    Scientific Publications, Boston. Studies in Microbiology; V.2:409    pp. (ISBN 0-632-01318-4).-   Sambrook, J. et al. eds., MOLECULAR CLONING: A LABORATORY MANUAL (2d    Ed. 1989) Cold Spring Harbor Laboratory Press, NY. Vols. 1-3. (ISBN    0-87969-309-6).-   SUSTAINED AND CONTROLLED RELEASE DRUG DELIVERY SYSTEMS, J. R.    Robinson, ed., Marcel Dekker, Inc., New York, 1978-   Winnacker, E. L. FROM GENES TO CLONES: INTRODUCTION TO GENE    TECHNOLOGY (1987) VCH Publishers, NY (translated by Horst    Ibelgaufts). 634 pp. (ISBN 0-89573-614-4).

EQUIVALENTS

The disclosure may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting of the disclosure. Scope of the disclosure is thusindicated by the appended claims rather than by the foregoingdescription, and all changes that come within the meaning and range ofequivalency of the claims are therefore intended to be embraced herein.

1. A modified oligonucleotide, comprising complementarity to a target, a sense and antisense strand with each strand comprising a 5′ end and a 3′ end, and at least one modified intersubunit linkage of Formula I:

wherein: each B is, independently, a base pairing moiety; W is selected from the group consisting of O, OCH₂, OCH, CH₂, and CH; each X is, independently, selected from the group consisting of halo, hydroxy, and C₁₋₆ alkoxy; Y is selected from the group consisting of O⁻, OH, OR, NH⁻, NH₂, S⁻, and SH; Z is selected from the group consisting of O and CH₂; when Y is O⁻ or S⁻, either Z or W is not O; R is a protecting group; and

is an optional double bond.
 2. The modified oligonucleotide of claim 1, wherein one or both of X is selected from the group consisting of OH OCH₃, and halo, optionally wherein the modified oligonucleotide does not comprise a 2′-fluoro substituent.
 3. The modified oligonucleotide of claim 1, wherein: Z is CH₂ and W is CH₂, optionally wherein the modified intersubunit linkage of Formula I is a modified intersubunit linkage of Formula II:

or Z is CH₂ and W is O, optionally wherein the modified intersubunit linkage of Formula I is a modified intersubunit linkage of Formula III:

or Z is O and W is CH₂, optionally wherein the modified intersubunit linkage of Formula I is a modified intersubunit linkage of Formula IV:

or Z is O and W is CH, optionally wherein the modified intersubunit linkage of Formula I is a modified intersubunit linkage of Formula V:

or Z is O and W is OCH₂, optionally wherein the modified intersubunit linkage of Formula I is a modified intersubunit linkage of Formula VI:

wherein: each X is independently, selected from the group consisting of fluoro, hydroxy, and C₁₋₆ alkoxy; Y is selected from the group consisting of O—, OH, and OR; Z is selected from the group consisting of O and CH₂ and W is OCH₂, and

is an optional double bond; or optionally wherein the modified intersubunit linkage of Formula I is a modified intersubunit linkage of Formula VIa:

or Z is CH₂ and W is CH, optionally wherein the modified intersubunit linkage of Formula I is a modified intersubunit linkage of Formula VII:

4-14. (canceled)
 15. The modified oligonucleotide of claim 1, wherein each base pairing moiety B is, independently, selected from the group consisting of adenine, guanine, cytosine, and uracil. 16-35. (canceled)
 36. The modified oligonucleotide of claim 1, wherein the modified intersubunit linkage is inserted on one or more of position 1-2; 5-6; 6-7; 10-11; 18-19; or 19-20 of the antisense strand. 37-40. (canceled)
 41. The modified oligonucleotide of claim 1, wherein the modified oligonucleotide is incorporated into siRNA, comprising a 5′ end, a 3′ end, and complementarity to a target, wherein the siRNA comprises a sense and antisense strand, and at least one modified intersubunit linkage of Formula I:

wherein: each B is, independently, a base pairing moiety; W is selected from the group consisting of O, OCH₂, OCH, CH₂, and CH; each X is, independently, selected from the group consisting of halo, hydroxy, and C₁₋₆ alkoxy; Y is selected from the group consisting of O⁻, OH, OR, NH⁻, NH₂, S⁻, and SH; Z is selected from the group consisting of O and CH₂; when Y is O⁻ or S⁻, either Z or W is not O; R is a protecting group; and

is an optional double bond.
 42. (canceled)
 43. The modified siRNA of claim 41, wherein: Z is CH₂ and W is CH₂, optionally wherein the modified intersubunit linkage of Formula I is a modified intersubunit linkage of Formula II:

or Z is CH₂ and W is O, optionally wherein the modified intersubunit linkage of Formula I is a modified intersubunit linkage of Formula III:

or Z is O and W is CH₂, optionally wherein the modified intersubunit linkage of Formula I is a modified intersubunit linkage of Formula IV:

or Z is O and W is CH, optionally wherein the modified intersubunit linkage of Formula I is a modified intersubunit linkage of Formula V:

or Z is O and W is OCH₂, optionally wherein the modified intersubunit linkage of Formula I is a modified intersubunit linkage of Formula VI:

wherein: each X is independently, selected from the group consisting of fluoro, hydroxy, and C₁₋₆ alkoxy; Y is selected from the group consisting of O⁻, OH, and OR; Z is selected from the group consisting of O and CH₂ and W is OCH₂, and

is an optional double bond; or optionally wherein the modified intersubunit linkage of Formula I is a modified intersubunit linkage of Formula VIa:

or Z is CH₂ and W is CH, optionally wherein the modified intersubunit linkage of Formula I is a modified intersubunit linkage of Formula VII:

44-54. (canceled)
 55. The modified siRNA of claim 41, wherein the base pairing moiety B is selected from the group consisting of adenine, guanine, cytosine, and uracil. 56-75. (canceled)
 76. The modified siRNA of claim 41, wherein the modified intersubunit linkage is inserted on one or more of position 1-2; 5-6: 6-7: 10-11: 18-19; or 19-20 of the antisense strand. 77-124. (canceled)
 125. The modified oligonucleotide of claim 1, wherein the modified oligonucleotide is incorporated into siRNA, said modified siRNA having a 5′ end, a 3′ end, that is complementary to a target and comprises a sense and antisense strand, wherein the siRNA comprises at least one modified intersubunit linkage is of Formula VIII:

wherein: D is selected from the group consisting of O, OCH₂, OCH, CH₂, and CH; C is selected from the group consisting of O⁻, OH, OR¹, NH⁻, NH₂, S⁻, and SH; A is selected from the group consisting of O and CH₂; R¹ is a protecting group;

is an optional double bond; the intersubunit is bridging two optionally modified nucleosides; and when C is O⁻ or S⁻, either A or D is not O.
 126. (canceled)
 127. The modified siRNA linkage of claim 125, wherein A is CH₂ and D is CH₂, optionally wherein the modified intersubunit linkage of Formula VIII is a modified intersubunit linkage of Formula IX:

or A is O and D is CH₂, optionally wherein the modified intersubunit linkage of Formula VIII is a modified intersubunit linkage of Formula XI:

or A is CH₂ and D is O, optionally wherein the modified intersubunit linkage of Formula VIII is a modified intersubunit linkage of Formula X:

or A is O and D is CH, optionally wherein the modified intersubunit linkage of Formula VIII is a modified intersubunit linkage of Formula XII:

or A is CH₂ and D is CH, optionally wherein the modified intersubunit linkage of Formula VIII is a modified intersubunit linkage of Formula XIV:

or A is O and D is OCH₂, optionally wherein the modified intersubunit linkage of Formula VII is a modified intersubunit linkage of Formula XIII:

128-138. (canceled)
 139. The modified siRNA linkage of claim 125, wherein each optionally modified nucleoside is independently, at each occurrence, selected from the group consisting of adenosine, guanosine, cytidine, and uridine, optionally wherein the modified nucleoside does not comprise a 2′-fluoro substituent. 140-153. (canceled)
 154. The modified siRNA linkage of claim 125, wherein the linkage is inserted at one or more of position 1-2; 5-6; 6-7; 10-11; 18-19; or 19-20 of the antisense strand. 155-158. (canceled)
 159. The modified oligonucleotide of claim 1, wherein R is a protecting group selected from the group consisting of dimethoxytrityl (DMTr), succinate, tert-butyl dimethylsilyl (TBDMS), benzoyl (Bz), benzyl (Bn), methoxyethoxymethyl ether (MOM), methoxybenzyl ether (PMB), methylthiomethyl ether, pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuranyl (THF), trityl (Trt), triisopropylsilyl (TIPS), tert-butyldiphenylsilyl (TBDPS), and acetate

is an optional double bond; and the intersubunit is bridging two optionally modified nucleosides. 160-196. (canceled)
 197. A branched compound comprising two or more oligonucleotides, wherein: (a) the oligonucleotides are connected to one another by one or more moieties selected from the group consisting of a linker, a spacer and a branching point, and (b) at least one oligonucleotide is a modified oligonucleotide comprising at least one modified intersubunit linkage of Formula (I):

wherein: each B is, independently, a base pairing moiety; W is selected from the group consisting of O, OCH₂, OCH, CH₂, and CH; each X is, independently, selected from the group consisting of halo, hydroxy, and C₁₋₆ alkoxy; Y is selected from the group consisting of O⁻, OH, OR, NH⁻, NH₂, S⁻, and SH; Z is selected from the group consisting of O and CH₂; when Y is O⁻ or S⁻, either Z or W is not O; R is a protecting group; and

is an optional double bond, optionally wherein the branched compound comprises 2, 4, 6, or 8 oligonucleotides.
 198. (canceled)
 199. The branched compound of claim 197, wherein each oligonucleotide is double-stranded and comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand each have a 5′ end and a 3′ end, optionally wherein: each double-stranded oligonucleotide is independently connected to a linker, spacer or branching point at the 3′ end or at the 5′ end of the sense strand or the antisense strand; each antisense strand independently comprises at least 16, at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides, and has complementarity to a target; and/or each linker is independently selected from the group consisting of an ethylene glycol chain, an alkyl chain, a peptide, an RNA, a DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, and combinations thereof; wherein any carbon or oxygen atom of the linker is: optionally replaced with a nitrogen atom, bears a hydroxyl substituent, or bears an oxo substituent. 200-203. (canceled)
 204. The branched compound of claim 197, wherein: Z is CH₂ and W is CH₂, optionally wherein the modified intersubunit linkage of Formula I is a modified intersubunit linkage of Formula II:

or Z is CH₂ and W is O, optionally wherein the modified intersubunit linkage of Formula I is a modified intersubunit linkage of Formula III:

or Z is O and W is CH₂, optionally wherein the modified intersubunit linkage of Formula I is a modified intersubunit linkage of Formula IV:

or Z is O and W is CH, optionally wherein the modified intersubunit linkage of Formula I is a modified intersubunit linkage of Formula V:

or Z is O and W is OCH₂, optionally wherein the modified intersubunit linkage of Formula I is a modified intersubunit linkage of Formula VI:

wherein: each X is independently, selected from the group consisting of fluoro, hydroxy, and C₁₋₆ alkoxy; Y is selected from the group consisting of O—, OH, and OR; Z is selected from the group consisting of O and CH₂ and W is OCH₂, and

is an optional double bond or optionally wherein the modified intersubunit linkage of Formula I is a modified intersubunit linkage of Formula VIa:

or Z is CH₂ and W is CH, optionally wherein the modified intersubunit linkage of Formula I is a modified intersubunit linkage of Formula VII:

205-215. (canceled)
 216. The branched compound of claim 197, wherein the base pairing moiety B is selected from the group consisting of adenine, guanine, cytosine, and uracil.
 217. The branched compound of claim 197, wherein the modified oligonucleotide is incorporated into a modified siRNA, said modified siRNA having a 5′ end, a 3′ end and complementarity to a target, wherein the siRNA comprises a sense and antisense strand, and at least one modified intersubunit linkage of Formula (I).
 218. The branched compound of claim 197, wherein R is a protecting group selected from the group consisting of dimethoxytrityl (DMTr), succinate, tert-butyl dimethylsilyl (TBDMS), benzoyl (Bz), benzyl (Bn), methoxyethoxymethyl ether (MOM), methoxybenzyl ether (PMB), methylthiomethyl ether, pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuranyl (THF), trityl (Trt), triisopropylsilyl (TIPS), tert-butyldiphenylsilyl (TBDPS), and acetate; and wherein

is an optional double bond.
 219. A compound of Formula (1): L-(N)_(n)   (1) wherein: L is selected from the group consisting of an ethylene glycol chain, an alkyl chain, a peptide, an RNA, a DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, and combinations thereof; wherein Formula (1) optionally further comprises one or more branch point Bp, and one or more spacer S; wherein: Bp is, independently for each occurrence, a polyvalent organic species ora derivative thereof; S is, independently for each occurrence, selected from the group consisting of an ethylene glycol chain, an alkyl chain, a peptide, an RNA, a DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, and combinations thereof, N is an RNA duplex comprising a sense strand and an antisense strand, wherein the sense strand and antisense strand each independently comprise one or more chemical modifications; and n is 2, 3, 4, 5, 6, 7 or 8; wherein at least one N comprises a modified intersubunit linkage of Formula (I):

wherein: each B is, independently, a base pairing moiety, optionally wherein the base pairing moiety B is selected from the group consisting of adenine, guanine, cytosine, and uracil; W is selected from the group consisting of O, OCH₂, OCH, CH₂, and CH; each X is, independently, selected from the group consisting of halo, hydroxy, and C₁₋₆ alkoxy; Y is selected from the group consisting of O⁻, OH, OR, NH⁻, NH₂, S⁻, and SH; Z is selected from the group consisting of O and CH₂; when Y is O⁻ or S⁻, either Z or W is not O; R is a protecting group; and

is an optional double bond.
 220. The compound of claim 219, wherein formula (1) comprises a structure selected from the group consisting of formulas (1-1)-(1-9):

optionally wherein: the antisense strand comprises a 5′ terminal group R selected from the group consisting of:

L has the structure of Li:

optionally wherein R is R³ and n is 2; or L has the structure of L2:

optionally wherein R is R³ and n is
 2. 221. (canceled)
 222. The compound of claim 219, comprising the structure of: Formula (2):

wherein: X, for each occurrence, independently, is selected from adenosine, guanosine, uridine, cytidine, and chemically-modified derivatives thereof; Y, for each occurrence, independently, is selected from adenosine, guanosine, uridine, cytidine, and chemically-modified derivatives thereof; - represents a phosphodiester internucleoside linkage; = represents a phosphorothioate internucleoside linkage; and --- represents, individually for each occurrence, a base-pairing interaction or a mismatch, wherein at least one of the - linkages or at least one of the = linkages of Formula (2) comprises a modified subunit linkage of Formula (I); or Formula (3):

wherein: X, for each occurrence, independently, is a nucleotide comprising a 2′-deoxy-2′-fluoro modification; and/or X, for each occurrence, independently, is a nucleotide comprising a 2′-O-methyl modification; and/or Y, for each occurrence, independently, is a nucleotide comprising a 2′-deoxy-2′-fluoro modification; and/or Y, for each occurrence, independently, is a nucleotide comprising a 2′-O-methyl modification, and wherein at least one of the - linkages or at least one of the = linkages of Formula (3) comprises a modified subunit linkage of Formula (I); or Formula (4):

wherein X, for each occurrence, independently, is selected from the group consisting of adenosine, guanosine, uridine, cytidine, and chemically-modified derivatives thereof; Y, for each occurrence, independently, is selected from the group consisting of adenosine, guanosine, uridine, cytidine, and chemically-modified derivatives thereof; - represents a phosphodiester internucleoside linkage: = represents a phosphorothioate internucleoside linkage; and --- represents, individually for each occurrence, a base-pairing interaction or a mismatch; wherein at least one of the - linkages or at least one of the = linkages of Formula (4) comprises a modified subunit linkage of Formula (I); or Formula (5):

wherein: X, for each occurrence, independently, is a nucleotide comprising a 2′-deoxy-2′-fluoro modification and/or X, for each occurrence, independently, is a nucleotide comprising a 2′-O-methyl modification and/or Y, for each occurrence, independently, is a nucleotide comprising a 2′-deoxy-2′-fluoro modification and/or Y, for each occurrence, independently, is a nucleotide comprising a 2′-O-methyl modification. 223-231. (canceled)
 232. The compound of claim 219, wherein Z is CH₂ and W is CH₂, optionally wherein the modified intersubunit linkage of Formula I is a modified intersubunit linkage of Formula II:

or Z is CH₂ and W is O, optionally wherein the modified intersubunit linkage of Formula I is a modified intersubunit linkage of Formula III:

Or Z is O and W is CH₂, optionally wherein the modified intersubunit linkage of Formula I is a modified intersubunit linkage of Formula IV:

or Z is O and W is CH, optionally wherein the modified intersubunit linkage of Formula I is a modified intersubunit linkage of Formula V:

or Z is O and W is OCH₂, optionally wherein the modified intersubunit linkage of Formula I is a modified intersubunit linkage of Formula VI:

wherein: each X is independently, selected from the group consisting of fluoro, hydroxy, and C₁₋₆ alkoxy; Y is selected from the group consisting of O—, OH, and OR; Z is selected from the group consisting of O and CH₂ and W is OCH₂, and

is an optional double bond; or optionally wherein the modified intersubunit linkage of Formula I is a modified intersubunit linkage of Formula VIa:

or Z is CH₂ and W is CH, optionally wherein the modified intersubunit linkage of Formula I is a modified intersubunit linkage of Formula VII:

233-243. (canceled)
 244. A delivery system for therapeutic nucleic acids having the structure of Formula (6): L-(cNA)_(n)   (6) wherein: L is selected from the group consisting of an ethylene glycol chain, an alkyl chain, a peptide, an RNA, a DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, and combinations thereof; wherein formula (6) optionally further comprises one or more branch point Bp, and one or more spacer S, wherein: Bp is independently for each occurrence a polyvalent organic species or derivative thereof; S is independently for each occurrence selected from the group consisting of an ethylene glycol chain, an alkyl chain, a peptide, an RNA, a DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, and combinations thereof, each cNA, independently, is a carrier nucleic acid comprising one or more chemical modifications; and n is 2, 3, 4, 5, 6, 7 or 8; and wherein at least one chemical modification of at least one cNA is an intersubunit linkage of Formula (I):

wherein: each B is, independently, a base pairing moiety; W is selected from the group consisting of O, OCH₂, OCH, CH₂, and CH; each X is, independently, selected from the group consisting of halo, hydroxy, and C₁₋₆ alkoxy; Y is selected from the group consisting of O⁻, OH, OR, NH⁻, NH₂, S⁻, and SH; Z is selected from the group consisting of O and CH₂; when Y is O⁻ or S⁻, either Z or W is not O; R is a protecting group; and

is an optional double bond.
 245. The delivery system of claim 244, wherein formula (6) comprises a structure selected from the group consisting of formulas (6-1)-(6-9):


246. The delivery system of claim 244, wherein the delivery system comprises one or more of the following: each cNA independently comprises at least 15 contiguous nucleotides; each cNA independently consists of chemically-modified nucleotides; the target of delivery is selected from the group consisting of brain, liver, skin, kidney, spleen, pancreas, colon, fat, lung, muscle, and thymus; and n therapeutic nucleic acids (NA), wherein each NA is hybridized to at least one cNA, optionally wherein: each NA independently comprises at least 16 contiguous nucleotides; or each NA independently comprises 16-20 contiguous nucleotides; or each NA comprises an unpaired overhang of at least 2 nucleotides, optionally wherein the nucleotides of the overhang are connected via phosphorothioate linkages; or each NA, independently, is selected from the group consisting of DNA, siRNAs, antagomiRs, miRNAs, gapmers, mixmers, or guide RNAs; or each NA is the same or not the same. 247-258. (canceled)
 259. The delivery system of claim 244, wherein: Z is CH₂ and W is CH₂, optionally wherein the modified intersubunit linkage of Formula (I) is a modified intersubunit linkage of Formula II:

or Z is CH₂ and W is O, optionally wherein the modified intersubunit linkage of Formula I is a modified intersubunit linkage of Formula III:

or Z is O and W is CH₂, optionally wherein the modified intersubunit linkage of Formula I is a modified intersubunit linkage of Formula IV:

or Z is O and W is CH, optionally wherein the modified intersubunit linkage of Formula I is a modified intersubunit linkage of Formula V:

or Z is O and W is OCH₂, optionally wherein the modified intersubunit linkage of Formula I is a modified intersubunit linkage of Formula VI:

wherein: each X is independently, selected from the group consisting of fluoro, hydroxy, and C₁₋₆ alkoxy; Y is selected from the group consisting of O—, OH, and OR; Z is selected from the group consisting of O and CH₂ and W is OCH₂, and

is an optional double bond; or optionally wherein the modified intersubunit linkage of Formula I is a modified intersubunit linkage of Formula VIa:

or Z is CH₂ and W is CH, optionally wherein the modified intersubunit linkage of Formula I is a modified intersubunit linkage of Formula VII:

260-271. (canceled)
 272. A double-stranded, chemically modified oligonucleotide comprising an antisense strand having complementarity to a target mRNA molecule and a sense strand having complementarity to the antisense strand, wherein the oligonucleotide comprises a modified intersubunit linkage represented by Formula I:

wherein: each B is, independently, a base pairing moiety; W is selected from the group consisting of —O—CH₂— and —O—CH═; each X is, independently, selected from the group consisting of halo, hydroxy, and C₁₋₆ alkoxy; Y is selected from the group consisting of O⁻, OH, OR, NH⁻, NH₂, S⁻, and SH; Z is selected from the group consisting of —O— and —CH₂—; R is a protecting group; and

is an optional double bond.
 273. The oligonucleotide of claim 272, wherein: each B is, independently, a base pairing moiety; W is selected from the group consisting of —O—CH₂— and —O—CH═; each X is, independently, selected from the group consisting of halo, hydroxy, and C₁₋₆ alkoxy; Y is selected from the group consisting of O⁻, OH, S⁻, and SH; Z is —O—; and

is an optional double bond.
 274. The oligonucleotide of claim 272, wherein the modified intersubunit linkage is represented by Formula VI:

wherein: each B is, independently, a base pairing moiety; each X is, independently, selected from the group consisting of fluoro, hydroxy, and C₁₋₆ alkoxy; Y is selected from the group consisting of O⁻, OH, OR, S⁻, and SH; Z is selected from the group consisting of —O— and —CH₂—; R is a protecting group; and

is an optional double bond.
 275. The oligonucleotide of claim 272, wherein the modified intersubunit linkage is represented by Formula VIa:

wherein: each B is, independently, a base pairing moiety; each X is, independently, selected from the group consisting of fluoro, hydroxy, and C₁₋₆ alkoxy; Y is selected from the group consisting of O⁻, OH, OR, S⁻, and SH; and R is a protecting group.
 276. The oligonucleotide of claim 272, wherein one instance of X is fluoro and the other instance of X is C₁₋₆ alkoxy.
 277. The oligonucleotide of claim 272, wherein the modified intersubunit linkage comprises a moiety represented by Formula XIII:

wherein: C is selected from the group consisting of O⁻, OH, OR, NH⁻, NH₂, S⁻, and SH.
 278. The oligonucleotide of claim 272, wherein each of the antisense strand and the sense strand is, independently, from 10 to 50 nucleotides in length, from 15 to 25 nucleotides in length, from 20 to 21 nucleotides in length, 15 or 16 nucleotides in length, 21 nucleotides in length, or 16 nucleotides in length.
 279. The oligonucleotide of claim 275, wherein each of the antisense strand and the sense strand is, independently, from 10 to 50 nucleotides in length, from 15 to 25 nucleotides in length, from 20 to 21 nucleotides in length, 15 or 16 nucleotides in length, 21 nucleotides in length, or 16 nucleotides in length.
 280. A double-stranded siRNA molecule comprising the oligonucleotide of claim
 272. 281. A branched compound comprising two or more oligonucleotides, wherein: (a) the oligonucleotides are connected to one another by one or more moieties selected from a linker, a spacer and a branching point, and (b) at least one oligonucleotide is a modified oligonucleotide comprising a modified intersubunit linkage represented by Formula (I):

wherein: each B is, independently, a base pairing moiety; W is selected from the group consisting of —O—CH₂— and —O—CH═; each X is, independently, selected from the group consisting of halo, hydroxy, and C₁₋₆ alkoxy; Y is selected from the group consisting of O⁻, OH, OR, NH⁻, NH₂, S⁻, and SH; Z is selected from the group consisting of —O— and —CH₂—; R is a protecting group; and

is an optional double bond.
 282. The branched compound of claim 281, wherein the modified intersubunit linkage is represented by Formula VI:

wherein: each B is, independently, a base pairing moiety; each X is, independently, selected from the group consisting of fluoro, hydroxy, and C₁₋₆ alkoxy; Y is selected from the group consisting of O⁻, OH, OR, S⁻, and SH; Z is selected from the group consisting of —O— and —CH₂—; R is a protecting group; and

is an optional double bond.
 283. The branched compound of claim 281, comprising two oligonucleotides connected to one another by way of a linker.
 284. The branched compound of claim 281, wherein each oligonucleotide is double-stranded and comprises an antisense strand having complementarity to a target mRNA molecule and a sense strand having complementarity to the antisense strand, and wherein each of the antisense strand and the sense strand is, independently, from 10 to 50 nucleotides in length.
 285. A compound represented by Formula (1): L-(N)_(n)   (1) wherein: L is selected from an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, and combinations thereof, wherein Formula (1) optionally further comprises one or more branch point Bp, and one or more spacer S, wherein Bp is independently for each occurrence a polyvalent organic species or derivative thereof; S is independently for each occurrence selected from an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, and combinations thereof; N is an RNA duplex comprising a sense strand and an antisense strand, wherein the sense strand and antisense strand each independently comprise one or more chemical modifications; and n is 2, 3, 4, 5, 6, 7 or 8, wherein at least one N includes a modified intersubunit linkage of Formula (I):

wherein: each B is, independently, a base pairing moiety; W is selected from the group consisting of —O—CH₂— and —O—CH═; each X is, independently, selected from the group consisting of halo, hydroxy, and C₁₋₆ alkoxy; Y is selected from the group consisting of O⁻, OH, OR, NH⁻, NH₂, S⁻, and SH; Z is selected from the group consisting of —O— and —CH₂—; R is a protecting group; and

is an optional double bond.
 286. A method of delivering an siRNA molecule to the central nervous system of a subject, the method comprising administering to the subject the siRNA molecule of claim
 285. 287. The method of claim 286, wherein the subject is a human.
 288. The method of claim 287, wherein the siRNA molecule is administered to the subject by way of intrathecal, intracerebroventricular, or intrastriatal injection. 